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  1. 1. Molecular Medicine Program Ιατρικη Σχολη Πανεπιστημιου Κρητης Diomedes E. Logothetis Membrane Structure and Proteins Ligand-gated Channels Lecture 2 November 14, 2007 The first part of the lecture will consider the membrane environment that ion channel proteins reside examining the lipid composition and forces that contribute to the thermodynamic stability of the lipid bilayer. A brief general introduction to membrane proteins and methods used to study them is included. The second part of the lecture will present ion channels that are gated by intracellular molecules (e.g. ATP, G proteins, cyclic nucleotides) as well as ion channels that are gated by extracellular molecules (e.g. the neurotransmitters acetylcholine and glutamate). Learning Objectives 1. Know the major classes of natural lipids, their structural characteristics and the properties of head groups and hydrocarbon chains 2. Understand the thermodynamic basis of lipid and detergent assembly: the hydrophobic effect 3. Understand the physical connection between the shape of lipid molecules and that of the aggregates they form: critical packing parameter 4. Understand the connection between the configuration of acyl chains, their packing and the properties of the bilayer 5. Know the connection between bilayer structure and dynamics and translational diffusion of lipids and proteins. Experimental approach to the measurement of diffusion of membrane components: Fluorescence Recovery After Photobleaching 6. Understand the characteristics of the fluid phase bilayer as shown by diffraction experiments and computer simulations of lipid dynamics 7. Modes of protein-membrane interaction. 8. Prediction of membrane protein structure and topology: Hydropathy analysis and the ‘positive-inside’ rule. 9. Lipid-modifications of proteins: hydrophobicity and membrane-binding affinity. 10. Modulation of reversible protein-membrane binding: the myristoyl switch. 11. Know the mechanism by which the metabolic state of the cell is coupled to membrane electrical events, such as those leading to secretion of insulin. 12. Know the mechanism of activation of G protein-gated K channels, as an example of a membrane-delimited pathway of regulating the activity of intracellular ligand-gated ion channels. 13. Modulation of Ion channels by soluble second messengers 14. Sensory transduction: Know the role of CNG channels in phototransduction. Understand how the balance of CNG and K channels gives rise to the quot;dark currentquot;, which is inhibited during a light flash. 15. Know the subunit composition of nicotinic ACh channels and general topology of the α subunits. 16. Know the general activation mechanism for NMDA and non-NMDA channels and role in LTP. 1
  2. 2. Readings: • Notes • Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P. Molecular Biology of the Cell. Fourth Edition, Garland Science. pp. 583-614 and pp. 631-657. • R. B. Gennis. Biomembranes: Molecular Structure and Function, Springer-Verlag, 1989 • Dr. Stephen H. White’s lab, at University of California at Irvine, maintains an interesting Web site you should visit: http://blanco.biomol.uci.edu 2
  3. 3. Membrane Structure Biological membranes are formed by two layers, or leaflets, of lipids. The outer surfaces of this bilayer are hydrophilic and exposed to water, whereas the interior of the bilayer is shielded from water and forms the hydrophobic core of the membrane. The main function of biological membranes is to act as permeability barriers, defining the inside and outside of the cell and delimiting functionally different compartments. This function alone, however, would not require the existence of the huge diversity of lipidic constituents found in nature. If the only function of membranes were to act as permeability barriers, it is reasonable to believe that just a few lipid species would be sufficient. Indeed, it has become evident that lipids perform a variety of functions in the physiology of the cell, some of which will be briefly discussed below. Lipids: chemical structures and classification Two features common to all lipids are a polar portion, or head group, which is exposed to the aqueous medium, and a non-polar portion, which is buried within the interior of the bilayer. 3
  4. 4. The major classes of lipids are: • Glycerophospholipids are the most abundant lipids, in which glycerol forms the common backbone of the molecule, with one of its hydroxyls linked to a phosphate via a phosphoester bond. The most common nomenclature used to describe the chemical structure of these lipids is the stereospecific numbering (sn) system. If the glycerol is drawn in a Fisher projection with the middle hydroxyl on the left, the three carbons are numbered as shown in the figure, and indicated as sn-1 for C(1), etc. 4
  5. 5. Glycerophospholipids include several subclasses: - 1,2-diacylphosphoglycerides or phospholipids, in which two of the glycerol hydroxyls (sn-1 and sn-2) are linked via ester bonds to fatty acid chains and the phosphate is at the sn-3 position. These are the most abundant lipids in eukaryotic and prokaryotic cells, excluding Archaebacteria, in which the phosphate is at the sn-1 position and the chains are linked to the glycerol via ether rather than ester bonds. - Lysophospholipids are phospholipids from which one of the acyl chains is missing. - Plasmalogens are phosphoglycerides in which one of the hydrocarbon chains is linked to glycerol via a vinyl-ether bond. Plasmalogens with an ethanolamine head group are abundant in myelin and in cardiac sarcoplasmic reticulum. - Cardiolipids are formed by the linkage of two phospholipids via phosphoester bonds to the two outer hydroxyls of a glycerol moiety, hence the alternative name of diphosphatidylglycerols. They are found in significant amounts in the inner membrane of mitochondria and in chloroplasts, but are rare in other membranes. • Glycoglycerolipids are lipids in which the sn-3 position of glycerol is linked to a carbohydrate via a glycosidic bond, rather than forming a phosphoester bond. 5
  6. 6. Monogalactosyldiacylglycerol has been named “the most abundant lipid in nature”, since it constitutes half of the thylakoid membrane in plant chloroplasts (the R groups in the figure represent the hydrocarbon chains of fatty acids). However, they are rare in animals. • Phosphosphingolipids, represented by sphingomyelin in the figure on p. 3, contain a backbone formed by sphingosine, rather than by glycerol. The details of the core structure of sphingolipids are illustrated in the diagram of the ganglioside GM1. Sphingosine is an amino alcohol in which the sn-2 OH is substituted by an amino group and a hydrocarbon chain is attached via a vinyl bond to the sn-1 carbon, leaving the sn-1 hydroxyl unreacted. A fatty acyl chain is linked to the sn-2 amino group via an amide bond, forming ceramide. As in glycerophospholipids, a phosphate bearing the head group is attached to the sn-3 position via a phosphoester bond. The amide and the hydroxyl group give these lipids the ability to form intermolecular hydrogen bonds, which may be significant in establishing interactions with proteins or in the formation of specialized membrane regions, or microdomains. • Glycosphingolipids are sphingolipids in which the sn-1 hydroxyl of ceramide is linked to a carbohydrate via a glycosidic bond. The carbohydrates, which constitute the 6
  7. 7. head groups of these lipids, vary from a single sugar to very complex polymers. Monogalactosyl ceramide is the most abundant component of the myelin sheath in nerve. Gangliosides contain oligosaccharides with one or more molecules of anionic sialic acid, while neutral oligosaccharides are contained in globosides. Glycosphingolipids are usually minor components of the outer leaflet of plasma membranes of animal cells, but they are significant in epithelial cells. The blood group antigens consist of glycosphingolipids on the surface of erythrocytes. • Sterols, represented by cholesterol in the figure above, are structurally separate from the previous lipid classes. The membrane-embedded portion of the molecule consists of a stiff ring structure and a short side chain, while the surface exposed portion is limited to a single OH group. The stiffness of the ring structure leads to changes in the dynamics and packing of the surrounding lipids. Cholesterol is found in animal cells, where it can contribute as much as 30% of the mass of lipid membranes, whereas plants contain other sterols. Head groups In addition to the carbohydrates already mentioned, a variety of polar head groups, differing in size, charge and chemical properties, are found in lipids. The structures of the most common head groups are shown in the context of a typical phospholipid, where the phosphate group is common to all head groups. 7
  8. 8. An unmodified phosphate represents the smallest head group, such as in phosphatidic acid, which carries a double negative charge at neutral pH. Addition of choline or ethanolamine, both positively charged at physiological pH, result in formation of the neutral head groups of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), two of the most common lipids. Additional negatively charged head groups are formed by modification of the phosphate with the zwitterionic amino acid serine, to give phosphatidylserine (PS), or with the uncharged glycerol or inositol moieties, yielding phosphatidylglycerol (PG) and phosphatidylinositol (PI), respectively. The individual charges contributed by the various lipid head groups are the major determinant of the overall electrostatic properties of biological membranes. Although only a minor constituent of cell membranes, phosphatidylinositol is the precursor of many important signal mediators generated by phosphorylation of several hydroxyls on the inositol by specific kinases. 8
  9. 9. Hydrocarbon chains An even greater variety exists in the types of hydrocarbon chains that are attached to lipids. The table below lists some of the more abundant acyl chains, including their common names and a useful condensed nomenclature. The first number indicates the number of carbons, or methylene units, in the chain, whereas the number on the right of the colon indicates its degree of unsaturation, i.e. the number of C=C bonds in the chain. For unsaturated chains, the isomeric form of the double bond, whether cis or trans, may also be given, as well as the number of the first carbon at which the double bond is located, sometimes preceded by a greek letter ∆. Acyl chains with even numbers of carbons are more abundant than those with odd numbers of carbons. The most common lengths are C16, C18 and C20, while the most common unsaturated chains are 18:1, 18:2, 18:3 and 20:4. The double bonds are usually in the cis configuration and in multiple unsaturated chains they are not conjugated, i.e. they are separated by at least two C-C bonds. A large fraction of 9
  10. 10. phospholipids have one saturated and one unsaturated chain, the latter usually linked to the sn-2 position of glycerol in animal cells. Thus, the number of possible combinations of head groups and acyl chains gives rise to a huge variety of lipid structures. As mentioned above, membranes within an organism do not contain uniform mixtures of these various lipids. Not only are cells in certain tissues enriched in specific types of lipids, but also different cellular organelles contain membranes formed by unique mixtures of lipids. Mitochondria, for example, are rich in cardiolipin and, indeed, the activity of cytochrome c oxidase, the component of the respiratory electron transfer chain responsible for the final step of oxygen reduction, is dependent on the presence of this lipid. Such fine-tuning of lipid composition extends even further, as demonstrated in the plasma membrane of human erythrocytes, in which the inner leaflet contains a different lipid mixture than the outer leaflet. Thus, with respect to neutral phospholipids, the extracellular leaflet is enriched in phosphatidylcholine and sphingomyelin, both bearing a phosphorylcholine head group, whereas phosphatidylethanolamine is localized preferentially in the inner leaflet. Most 10
  11. 11. striking, though, is the absolute exclusion from the outer leaflet of the anionic phospholipid phosphatidylserine. Such asymmetric distribution of amino-phospholipids is believed to be maintained by ATP-requiring enzymes, called translocases, which catalyze the transbilayer transport of these lipids. The physiological function of the surface expression of phosphatidylserine is related to the clearance of aged red cells. After release into the bloodstream, a red cell remains in circulation for 120 days on average. During this time, oxidative stress progressively deteriorates the biochemical machinery of the cell, including the enzymatic activities responsible for the segregation of this lipid. Eventually, phosphatidylserine appears on the outer surface of the erythrocyte, and this signal is recognized by phagocytic cells within the spleen, which clear the aged cell from circulation. Platelets constitute another system in which the asymmetric phosphatidylserine distribution is physiologically significant. In this case, the anionic lipid, which is normally absent from the surface, becomes expressed on the outer membrane leaflet upon activation of platelets at a site of injury. Together with calcium, phosphatidylserine is an essential activator of blood coagulation factors. Under physiological conditions, as well as in culture, cells may undergo apoptosis, a controlled process of suicide. Thus, during development of the immune system, self-recognizing thymocytes are eliminated in order to prevent autoimmune reactions. One of the signatures of an apoptotic cell, in addition to DNA degradation, is the expression of phosphatidylserine on the extracellular surface. This observation has been used to develop an apoptosis test, which makes use of fluorescence-labeled 11
  12. 12. recombinant annexin, a normally cytoplasmic protein that binds specifically to phosphatidylserine via calcium bridges. Phospholipases and lipids as precursors of second messengers Another essential function of lipids is as precursors of many second messengers that participate in a variety of signaling pathways. Each of these molecules is generated by cleavage of a lipid precursor at a specific bond. For this purpose, several classes of enzymes, named phospholipases, exist. Phospholipase A2 enzymes hydrolyze the ester bond between glycerol and the sn-2 acyl chain of phospholipids, thus generating a lysophospholipid and a free fatty acid. Among the released fatty acids is arachidonic acid, which is oxidized to other active metabolites, such as prostaglandins, which are involved in inflammation and other patho- physiological processes. Analogous enzymes, called phospholipase A1, hydrolyze the ester bond at the sn-1 position, but they are not yet well characterized. Phospholipase C catalyzes the hydrolysis of the phosphoester bond, releasing a soluble phosphorylated head group and a diacylglycerol. Of widespread significance is the cleavage of phosphatidylinositol(4,5)-bisphosphate (PIP2), which generates two second messengers: the water-soluble inositol(1,4,5) trisphosphate (IP3), which causes the release of calcium from the endoplasmic reticulum by binding to the IP 3 receptor found in the membrane of this organelle, and the membrane-bound 1-stearyl-2- 12
  13. 13. arachidonyl diacylglycerol, which is responsible for activation of protein kinase C and enhanced phosphorylation of downstream signaling proteins. Phospholipase D instead generates phosphatidic acid and a free head group. Phosphatidic acid is becoming recognized as a second messenger, for example as an activator of the NADPH oxidase responsible for the generation of reactive oxygen products in neutrophils activated upon binding to immunoglobulin-coated bacteria. Hydrophobicity and thermodynamics of lipid assembly In this section, we will try to answer semi-quantitatively the following questions: Which intermolecular forces are involved in the assembly of lipid membranes? What is the thermodynamic basis for the formation and stability of the bilayer? Three basic forces contribute to the stability of lipid aggregates: The van der Waals force, which is a short-range electrostatic interaction between instantaneous dipoles on adjacent molecules. This interaction develops as a result of the formation of a dipolar charge in one molecule, due to fluctuations of its electronic-nuclear distribution, and the instantaneous induction of a dipole of opposite orientation on an adjacent molecule. This dipole-induced-dipole interaction stabilizes the overall interaction between the two molecules and is proportional to the intermolecular contact surface. These van der Waals interactions occur among all types of molecules, between head groups at the water-bilayer interface as well as between hydrocarbon chains in the interior of the membrane. The electrostatic force is also responsible for the stronger ionic interaction between charged groups and for the formation of hydrogen bonds between hydrogen- 13
  14. 14. bond donors, such as NH and OH, and acceptors, such as CO, on adjacent sphingolipids. Hydrogen bonding is particularly extensive between water molecules and is the origin of many of its solvent properties, such as its dielectric constant. Attractive intermolecular ionic interactions form between the head groups of zwitterionic lipids, such as phosphaditylcholine, whereby the negatively charged phosphate on one head group interacts with the positively charged ammonium ion of the choline on a nearby head group. However, the greatest contribution to the stability of the membrane bilayer comes from the hydrophobic force, or hydrophobic effect. Its physical origin can be understood as follows. A hydrophobic molecule, such as a hydrocarbon chain, placed in water must be surrounded by water molecules interacting with it. However, whereas water-water interactions are stabilized by intermolecular hydrogen bonds, these favorable polar interactions cannot be established between water and a molecule that does not have hydrogen-bond donor or acceptor groups. Therefore, although bound water molecules still maintain most of their hydrogen bonds with free water molecules, some favorable electrostatic interactions are lost when water binds to the hydrocarbon chain. This loss of hydrogen bonds represents an energetic, or enthalpic, penalty for the system. However, the entropic cost of immobilizing water molecules on the surface of the hydrocarbon chain is much more significant than this enthalpic penalty. The water molecules bound to the hydrophobic surface lose the rotational and translational degrees of freedom they had in pure liquid water. Because the free energy of the system, ∆G, is given by ∆G = ∆H − T ∆S 14
  15. 15. where ∆H and ∆S represent the contributions of enthalpy and entropy, respectively, the reduction in the ∆S term due to immobilization of water on the hydrophobic surface leads to an increase in the ∆G of the system. For analogous reasons, placing water molecules within the hydrophobic core of the membrane is also very unfavorable. However, if separate hydrocarbon chains in water aggregate and pack tightly, the water molecules immobilized on their surfaces can be released back into the bulk, thus regaining their lost degrees of freedom. This leads to an overall increase in the ∆S term and a reduction in the ∆G of the system. Thus, the thermodynamic basis for the stability of the lipid bilayer lies in the entropy difference between a system consisting of isolated hydrocarbon chains coated with immobilized water molecules and a system in which aggregation of hydrocarbon chains and formation of a hydrophobic bilayer phase lead to release of solvation water back to the bulk aqueous phase. Additional stabilization arises from the enthalpic contributions of van der Waals interactions between lipid chains as well as ionic and H-bonding interactions between lipid head groups. The hydrophobicity of a molecule can be determined quantitatively by measuring its distribution, or partition, in a solvent system composed of water and an immiscible hydrocarbon phase, such as hexane. The equilibrium constant, or partition coefficient, K of the solute in this system is defined as: [ X] H O K= 2 [ X] HC where the [X] values represent the solute concentration in mole fraction units in water and in the hydrocarbon phase, respectively. The partition coefficient K is related to the 15
  16. 16. standard state free energy of transfer, ∆G0trans, of the solute from water to the hydrocarbon phase ∆G 0 = µ 0 2O − µ 0 = − RT ln K trans H HC where the µ0 values represent the standard chemical potentials of the solute in water and in the hydrocarbon phase, respectively, R is the gas constant (1.987 cal °K -1 mol-1) and T is the absolute temperature (°K). It turns out that the hydrophobicity, as measured by ∆G0trans, is proportional to the surface of contact between the hydrophobic solute and water, which determines the number of water molecules that would be constrained. The proportionality constant for transfer of an alkane chain from water into a hydrocarbon phase is found to be ∆G0trans ≈ −25 cal / Å2. Based on surface area, each additional methylene (CH2) group contributes ~ −800 cal/mol to the hydrophobicity of the chain. At 25°C, this contribution increases the partition coefficient K of the alkane chain by a factor of ~4 in favor of the hydrocarbon solvent. The partition coefficient of water in hexadecane (C16) indicates that the water concentration within the membrane is in the millimolar range, corresponding to about 1 water molecule per 1,000 phospholipid. Detergents and micelle formation A short-chain alkane, at very low concentration, can be dissolved in pure water. However, as more alkane is added, a concentration is reached at which a separate phase forms, into which any additional alkane partitions. This critical concentration is called the solubility limit. 16
  17. 17. Detergents and lipids, however, are amphiphilic molecules. For example, sodium dodecyl sulfate (SDS), a detergent commonly used as a denaturant for protein gel electrophoresis, has a 12-carbon long chain terminated by a charged sulfate group. Up to a critical concentration of ≈ 1-2 mM (in 0.1M Na+), SDS dissolves in monomeric form. Upon reaching the critical concentration, however, it forms a new phase composed of Each SDS micelle contains ≈100 monomers, spherical aggregates called micelles. whose hydrophilic head groups delimit the water-exposed surface while their methylene chains form the “oily” interior. Additional SDS increases the concentration of micelles, while the concentration of monomers in solution remains constant. The critical concentration in this case is called the critical micellar concentration (CMC). Since the CMC is a measure of hydrophobicity, its value is a function of the chemical structure of the amphiphile. For example, detergents with longer chains have lower CMCs, as shown by decylmaltoside (10-C long, CMC=1.5mM) versus dodecylmaltoside (12-C long, CMC=0.12mM). Biological phospholipids, with two long methylene chains, have extremely low CMCs, below 10−10 M. For this reason, exchange of lipids between membranes is 17
  18. 18. facilitated by water-soluble cytosolic proteins, called phospholipid transfer proteins, which bind lipid monomers and carry out the one-for-one exchange. Lipid structure, lipid shape and lipid aggregate shape The high-resolution structures of several lipids have been determined by x-ray diffraction, using lipid crystal containing very little hydration water. A few examples are given in the figure below. A few noticeable features of these structures are: • Within the crystals, the lipid are found in a lamellar arrangement, with the hydrophilic and hydrophobic groups organized in stacked bilayers 18
  19. 19. • The acyl chains are fully extended, or in the all-trans configuration, with the exception of the first 2 methylenes in the sn-2 chains of PC and PE, which are oriented parallel to the crystallographic bilayer plane • The tilt of the acyl chains away from the normal to the bilayer surface increases from PE, in which they are virtually perpendicular, to PC , where the angle is ≈12°, to the cerebroside, where the angle is 41°. • The glycerol is oriented perpendicular to the bilayer plane, whereas the choline and ethanolamine head groups are almost parallel to the plane. In fact, the amino group of ethanolamine interacts with the unesterified oxygens of an adjacent molecule A closer examination of the structural parameters of a PC molecule allows us to understand the reason for the differences in packing and acyl chain orientations among the various lipids. The volume occupied by the molecule can be divided into two parts: a polar region, which includes the head group and the glycerol, and a hydrophobic region, comprising the two acyl chains, except the first two carbons of the sn-2 chain. Each region also defines an area given by its projection onto the plane of the membrane: the head group cross- 19
  20. 20. section area, S, and the acyl chain area, 2Σ, where Σ is the cross-section area of each acyl chain. For saturated chains in the all-trans configuration, Σ=19Å2. The relationship between these two areas determines the degree of tilt of the acyl chains in the crystals, as well as the shape of the aggregates formed by the lipid in aqueous solution. If 2Σ < S, the chains will tilt to maximize the intermolecular van der Waals contacts and accommodate the larger head group. This occurs in PC, where choline occupies an area S≈50Å2. On the other hand, in PE S≈39Å2, so that 2Σ ≈ S and the chains can attain optimal packing without tilting. These qualitative considerations can be formulated in a more quantitative, albeit empirical, formalism that allows us to predict the shape of the aggregate that each lipid forms in solution. This formulation is based on the concept of the critical packing parameter, which is defined as v / lSo, where v is the volume of the hydrocarbon portion of the molecule, l is the maximum length of the acyl chain, and So is the optimal surface area occupied by the molecule at the interface between the aggregate and water. So is determined by the balance of repulsive and attractive interactions between head groups, and is sensitive to solution conditions, such as ionic strength, divalent cations and pH. The ratio v/l is analogous to the cross-sectional area of the hydrocarbon portion (≈2Σ). The molecular shapes of amphiphilic molecules corresponding to each value of the critical packing parameter are illustrated in the figure below, together with the shape of the aggregate they form in solution. 20
  21. 21. Phospholipids, in general, have a good match between the areas of the two molecular regions, and therefore they tend to naturally assemble in planar membranes, forming a so-called bilayer phase. Under certain conditions, however, some of them tend to form non-bilayer structures, consisting of tubular assemblies in which the head groups face the interior lumen filled with water while the acyl chains, oriented outwards, contact the chains from nearby cylinders. This macroscopic structure is called an inverted hexagonal phase, HII, because the hydrophobic cylinders pack in a hexagonal pattern. Lysophospholipids and most detergents, in which the single hydrocarbon chain has a much smaller cross-sectional area than their head groups, form spherical assemblies and give rise to a micellar phase. Both of these lipid phases are thought to form locally at sites of membrane fusion. 21
  22. 22. Fluid-bilayer structure X–ray diffraction structures of lipid crystals show the bilayers as planar and well- ordered structures, with all-trans acyl chains. Similarly, lower resolution TEM images convey the impression that biological membranes are flat slabs, in which the lipid head groups form smooth, planar surfaces. However, fluid-phase lipid bilayers are very different from these pictures. The ‘structure’ of fluid bilayers has been determined by x- ray and neutron diffraction methods using multilamellar stacks of bilayers, which present a periodic order in the direction perpendicular to the membrane plane. This one- dimensional order allows the measurement of the distribution of matter along the bilayer normal. Thus, the ‘structure’ of a fluid bilayer represents the time-averaged spatial distribution of structural groups of the lipid (carbonyls, phosphates, double bonds, etc.) projected onto the axis perpendicular to the bilayer plane. These distributions give the probability of finding a particular structural group at a specific location along the axis, which represents the distance from the center of the hydrophobic core of the bilayer. The structure of a fluid bilayer of dioleoylphosphatidyl- 22
  23. 23. choline (DOPC, di-C18:1 cis-∆9) is shown in panel (b). The gaussian peaks give an accurate representation of the true thermal motion of the molecules, which is a fundamental property of fluid bilayers that plays a critical role in the interaction of peptides and proteins with lipid membranes. Several features of the fluid DOPC bilayer are important. First, the great amount of thermal disorder is revealed by the widths of the probability density peaks, as illustrated by the ≈10Å widths of the PO4 in the head groups and of the C=C acyl chain group. The width of the C=C group is a vivid graphical representation of the effect of the random configurational rearrangements occurring within the acyl chains, which originate from thermally activated trans-gauche bond isomerizations. Second, the overall thermal thickness of the interfacial region, defined by the distribution of the water of hydration, is ≈30 Å, equal to that of the hydrocarbon core of the bilayer. As illustrated by the end view diagram in panel (b) of the figure above, a peptide in an α-helical conformation has a cross-sectional diameter of ~10 Å and can be easily accommodated within the 15 Å thickness of the interface. Third, the physico-chemical environment in the interface region is highly heterogeneous, presenting a steep but not abrupt change from the solvation properties of water to those of the membrane hydrocarbon core. Many membrane-supported reactions and protein-protein or lipid-protein interactions occur in this heterogeneous environment. As illustrated in panel (a) of the figure, a transmembrane α-helical peptide of twenty amino acids can be completely accommodated within the hydrophobic core of the bilayer, whereas longer peptides will 23
  24. 24. have their ends protruding into the interfacial region. This imposes constraints on the allowed amino acid sequences of the peptide, as discussed later. These conclusions drawn from the two-dimensional representation of the fluid bilayer structure are fully confirmed by the three-dimensional pictures of bilayers obtained by computer simulations of the molecular dynamics of lipid bilayers. A snapshot of the transversal cross section of the bilayer extracted from one of these simulations is shown in the figure below. 24
  25. 25. The acyl chains, represented by the gray lines, are very disordered, each with several kinks introduced by gauche bond configurations and only short all-trans segments. The head group atoms, shown in red, are distributed over a wide range of depths, and the surface delimiting the head-group region from the hydrocarbon region is very rough. In agreement with the neutron diffraction data, water molecules, represented by the blue oxygen atoms and white hydrogen atoms, are found deep in the interfacial region, indeed as far as the boundary of the hydrophobic core. Despite the disorder and the high degree of conformational fluctuations, the acyl chains maintain tight packing and good van der Waals contacts, in agreement with the minor increase in specific volume at Tm. Intrachain motions and translational diffusion of individual molecules occur via thermally induced structural and packing fluctuations of the surrounding molecules. This dense and crowded hydrophobic environment is the origin of the high performance of thin biological membranes as permeability barriers, as well as of the high cooperativity of lipid phase transitions. Based on these experimental and theoretical results, the common illustrations, which depict bilayers as two smooth surfaces separating polar and apolar regions, must be considered misleading. 25
  26. 26. Membrane Proteins: Structure and Interactions Prediction of protein topology Identification of protein transmembrane domains – Sequence hydropathy analysis. The difficulties of membrane protein crystallization and structure determination, on the one hand, and the abundance of sequence information, on the other, have led to efforts to develop theoretical methods for the prediction of the location of potential transmembrane domains. Perhaps the most widely used of such methods is one based on sequence hydropathy analysis. The first step in such analysis is to evaluate the degree of hydrophilicity or hydrophobicity of the protein along its amino acid sequence. Because of the hydrophobic environment in the bilayer core, a membrane-spanning protein segment is expected to contain a preponderance of apolar amino acids. The transmembrane sequence should also be folded, either as an α helix or as a β strand in a β barrel, so that peptide H-bonds are satisfied intramolecularly rather than by bringing bound water into the hydrophobic bilayer core. Thus, the method attempts to find the location and number of transmembrane segments based on the relative hydrophilic and hydrophobic properties of contiguous stretches of the amino acid sequence. For this purpose, hydropathy scales have been devised to rank the relative hydrophilicity and hydrophobicity of the 20 amino acids. One of these scales, proposed by Kyte and Doolittle in 1982 (J. Mol. Biol. 157: 105-132), is still in widespread use. 26
  27. 27. This scale was defined using the water-vapor partition coefficient or standard free energy of transfer, ∆G0transfer, of the amino acid side chains as well as their degree of surface exposure in proteins of known crystallographic structure. The hydropathy indexes are normalized between 4.5 and −4.5, with positive values indicating that free energy is required to transfer the side chain to water and the amino acid is considered hydrophobic. Conversely, negative values indicate that free energy is released upon transferring the side chain into water and the amino acid is hydrophilic. Given an amino acid sequence, the residue letter code is substituted with its corresponding hydropathy index to give the sequence hydropathy profile. A window of odd length, usually 7–13 residues, is scanned along the sequence. The hydropathy index values within the window are summed, and the average hydropathy value of the segment is computed by dividing the sum by the size of the window, as shown below. 27
  28. 28. E I T W I V G M V I Y L L M M G A i= 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 |−−−−−− sliding window −−−−−−| Hi= -3.5 4.5 -0.7 -0.9 4.5 4.2 -0.4 1.9 4.2 4.5 -1.3 3.8 3.8 1.9 1.9 -0.4 1.8 Using a window size w = 7, the first value of the sequence hydropathy profile, Dl, corresponds to position 4. The general recursive formula for the computation of Dl is i =l + k ∑H i k = ( w − 1) 2 Dl = i =l − k w D4 = (-3.5+4.5-0.7-0.9+4.5+4.2-0.4) / 7 = 1.1 D5 = (4.5-0.7-0.9+4.5+4.2-0.4+1.9) / 7 = 1.87 Finally, a threshold value for the segmental hydropathy must be selected to decide when a stretch of amino acids may be considered hydrophobic enough to be a potential transmembrane protein sequence. For the Kyte-Doolittle scale, this threshold value is usually taken to be 1.0-1.25. Since the thickness of the bilayer hydrophobic core is ~30Å and each amino acid contributes 1.5Å to the length of the α helix, a potential transmembrane segment in this conformation is expected to be at least 20-residue long. Thus, a putative α-helical transmembrane segment should be identifiable by a consecutive stretch of ~20 values of Dl above the hydropathy threshold value. As an example, the figure below shows the plot of the hydropathy profile of glycophorin A, as it appeared in the original Kyte and Doolittle paper. 28
  29. 29. A sliding window of 7 residues was used to compute each value of hydropathy index. However, the authors reported the sum of the indexes for the residues within the sliding window rather than the average value, as it is now common practice (i.e. the hydropathy index in the figure is 7×Dl). The single α-helical transmembrane segment is easily identified in the region of residues 73-95. The “positive inside” rule Once the putative transmembrane segments have been identified, their orientation in the membrane must be chosen. Statistical analysis of single- and multi- spanning proteins indicated that their topological determinants may reside in the polar and loop regions flanking the transmembrane segments. In particular, the distribution of positively charged residues, arginine and lysine, was well correlated with the topology. In a sample of bacterial inner membrane proteins, the frequency of Arg + Lys residues was 4-fold higher in the cytoplasmic than in the periplasmic membrane flanking regions. A similar bias, though not as strong, has been found to apply to proteins from higher organisms as well. Thus, the “positive inside” rule, first proposed by G. von Heijne, is thought to be a fundamental determinant of the topology of most integral membrane proteins. Clusters 29
  30. 30. of positively charged amino acids found near one end of a predicted transmembrane α helix, identify it as the cytoplasmic end. Lipid-anchored proteins Intrinsic proteins are anchored to membranes of eukaryotic cell not only via transmembrane domains, but also via covalently attached hydrocarbon chains. Four classes of lipid-anchored proteins are distinguished by the type of chain and linkage. 1. Glycosylphosphatidylinositol (GPI)-anchored proteins are a heterogeneous family of proteins found on the exoplasmic surface of cell membranes. The GPI anchor is formed by a phosphatidylinositol linked, by N-acetylglucosamine, to a polymannose chain and a phosphoethanolamine. The linkage between protein and anchor is always located at the carboxyl-terminal amino acid. The GPI anchor is always added post-translationally after proteolytic removal of a peptide, 17-31 residues long, from the C-terminus of a protein precursor. GPI-anchored proteins include hydrolytic enzymes, (alkaline phosphatase, acetylcholine esterase, 5’- nucleotidase), adhesion proteins (neural cell adhesion molecule or N-CAM), receptors (FcγRIIIB, a neutrophil receptor for Fc region of immunoglobulin G). GPI-anchored proteins can be released from the surface by treatment with exogenous phosphatidylinositol-specific phospholipase C. 2. N-Myristoylated proteins are found anchored to the cytoplasmic face of the plasma membrane and to the membranes of other organelles. The 14-carbon chain is attached co-translationally via a stable amide linkage to an N-terminal glycine residue. Examples of N-myristoylated proteins are the protein tyrosine kinase p60src and the catalytic subunit of cAMP-dependent protein kinase, also known as PKA. 30
  31. 31. 3. S-prenylated proteins are anchored to the cytoplasmic plasma membrane surface by a 15-carbon farnesyl or a 20-carbon geranylgeranyl unsaturated chain. The thio- ether linkage of the anchor to a cysteine residue in the protein is catalyzed by a farnesyl-transferase. The cysteine is initially the fourth residue from the C-terminus of the protein. However, after the chain is attached, the 3 C-terminal residues are cleaved by a protease and the carboxyl group of the cysteine is methylated. 4. S-palmitoylated proteins are anchored to the plasma membrane by a 16-carbon saturated palmitic chain via a thio-ester linkage. This modification may occur by spontaneous reaction of palmitoyl-coenzyme A with a protein already associated with the membrane or may be catalyzed by a membrane-associated palmitoyl- transferase. S-palmitoylation can take place on exposed cysteines anywhere in the protein. Contrary to thio-ether bonds, a thio-ester linkage is labile and is hydrolyzed under mild basic conditions. Proteins known to be palmitoylated include p21ras, the glycoprotein hemagglutinin of influenza virus, the mammalian transferrin receptor 31
  32. 32. and the visual receptor rhodopsin, which is a seven-helix transmembrane protein. In many cases, doubly acylated proteins are generated first by co-translational N-myristoylation and, after interacting with the membrane, by S-palmitoylation. The protein affinity for the membrane conferred by these lipid anchors has been estimated by studies of the equilibrium binding of lipid-modified fluorescent peptides. The results are summarized in the table below. On the right column, the values of the effective dissociation constant, Kdeff, are listed. The binding energy of fatty acids and acylated peptides to phospholipid bilayers increases linearly with the number of carbons in the chain. The slope of 0.8 kcal mol-1 per -CH2 group is equal to that found for the partitioning of the neutral form of a fatty acid from water into a bulk alkane phase. The values of Kdeff for myristoyl and palmitoyl anchors reflect the increase in ∆G0transfer upon addition of two methylenes. Thus, the binding energy of acylated peptides and proteins to membranes is due to the classical hydrophobic effect. The membrane affinities of whole proteins are estimated to be about 10-fold lower than those for short peptides, in other words their Kdeff values are about tenfold higher. Thus, a myristoylated protein is expected to have a Kdeff ≈0.8mM 32
  33. 33. while the concentration of lipids in a cell is approximately millimolar. Since the lipid concentration is comparable to Kdeff, simple myristoylation provides barely enough hydrophobic energy to attach a protein to a phospholipid bilayer. Other factors, such as electrostatic interactions between amino acid side chains and phospholipid head groups, may help in partitioning these proteins to the membrane. These relatively weak membrane-protein affinities create the potential for dynamic and reversible membrane localization of these proteins, which may be targets of metabolic modifications leading to changes in their binding affinity and effective concentration at the membrane. Modulation of reversible protein-membrane interactions: The myristoyl- electrostatic switch An example of an electrostatic charge-induced reversible membrane association is given by the myristoylated protein MARCKS, an acronym for myristoyl alanine-rich C- kinase substrate. MARCKS binds to Ca2+-calmodulin and actin and is thought to integrate protein kinase C (PKC) and Ca2+-calmodulin signals that affect interactions of actin with the cytoskeleton and membranes. Binding of calmodulin requires Ca2+ and is prevented by PKC phosphorylation of MARCKS, whereas binding and crosslinking of actin filaments by MARCKS is blocked by phosphorylation and by Ca2+-calmodulin. MARCKS is a rod-shaped protein with at least two domains: an N-terminal myristoylation domain and a basic effector domain that contains the PKC phosphorylation sites as well as the calmodulin and actin-binding sites. Three peptide fragments, corresponding to the basic domain of MARCKS, were used to characterize the effect of changes in their electrostatic properties on membrane affinity: MARCKS 151-175: KKKKKRFSKKSFKLSGFSFKKNKK Tetra-Asp MARCKS: KKKKKRFDKKDFKLDGFDFKKNKK Phos-MARCKS: KKKKKRFS(P)KKS(P)FKLS(P)GFS(P)FKKNKK 33
  34. 34. The MARCKS peptide is strongly cationic, because of all the lysine residues, whereas the positive charges in the Asp-substituted and the tetra-phosphorylated peptides are progressively neutralized by the added negative groups. Binding of the peptides was measured as a function of the fraction of phosphatidylserine in the bilayer. As shown in the figure below, addition of negative charges to the peptide diminishes its affinity for the membrane. Thus, a higher density of anionic head groups is required to reach 50% binding to the bilayer. Phosphorylation is particularly effective. Thus, in the presence of bilayers containing 10-20% acidic lipids, MARCKS phosphorylation leads to almost complete desorption of the peptide from the membrane. This effect was demonstrated in a kinetic experiment by using mixed bilayers containing phosphatidylserine and fluorescence-labeled tracer lipids. Under the experimental conditions, the fluorescence intensity was proportional to the degree of peptide binding to the membrane. Addition of MARCKS 151-175 peptide produced an increase in the fluorescence of the labeled lipids. Upon addition of PKC in the presence of ATP, serine phosphorylation caused the immediate desorption of the peptide from the 34
  35. 35. membrane, which was completed after ~1 min, as judged by the decrease of the fluorescence signal back to the level measured in the absence of peptide. The binding of full-length MARCKS to membranes requires the contribution of both the hydrophobic interactions of the myristoyl anchor with the bilayer core and the electrostatic interactions between the basic domain and anionic lipid head groups. Phosphorylation by PKC reduces the electrostatic binding energy and leads to desorption of MARCKS, until dephosphorylation by protein phosphatases restores the full interaction energy. Thus, the myristoyl anchor facilitates the initial transfer of the protein to the membrane, which then increases the chance that the basic domain will associate electrostatically with the anionic lipids. This mechanism for reversible protein- membrane binding is known as the myristoyl-electrostatic switch. Membrane anchoring of peripheral proteins via non-covalently bound lipids Protein targeting to specific cellular compartments in response to external stimuli is a fundamental component of signal transduction mechanisms in eukaryotic cells. Such localization can be achieved by means of protein-protein interaction domains, such as src-homology 2 (SH2) and src-homology 3 (SH3) domains, which recognize specific phosphotyrosine motifs and proline-rich sequences, respectively, in the protein binding partners (e.g. activated receptors). Alternatively, localization can be carried out by protein domains that bind specifically to lipids embedded in cell membranes. The best known members of this class of domains are the protein kinase C (PKC) homology-1 (C1) and PKC homology– 2 (C2) domains, the FYVE domain, and the pleckstrin homology (PH) domain. Some C1, C2, and PH domains interact with proteins in addition to or instead of lipids. Fluorescence microscopy and fusion proteins derived from green fluorescent protein (GFP) and several of these domains have allowed detailed studies of the kinetics of spatial redistribution (e.g. from cytosol to membranes or vice versa) during signaling. 35
  36. 36. A model of the membrane-docked structure of a representative domain of each type is shown in the figure below. Panel A: structure of the complex between the C1 domain of PKCδ and phorbol ester is shown with the model of a myristoyl chain. Panel B: structure of the Ca2+-bound C2 domain of cPLA2 interacting with the membrane. Panel C: structure of the FYVE domain of Vps27p with a model of phosphatidylinositol 3-phosphate (PI3P). Panel D: 36
  37. 37. structure of the PH domain of PLCδ1 complexed with Ins(1,4,5)P3 and with a model of the two myristoyl chains. The secondary structure and molecular surface of each domain are shown. The surface colors indicate the nature of the residues, green for hydrophobic and blue for basic. Some specific and nonspecific contact residues are also indicated. The two Zn2+ in the C1 and FYVE domains are shown as cyan circles, while the two Ca2+ in the C2 domain are shown as blue circles. The domains are positioned so that known membrane-interacting residues penetrate the membrane and patches of basic residues are near the membrane surface. The membrane leaflet, drawn to scale, is divided into an interfacial zone and a hydrophobic core, each ~15 Å thick. C1 domains Originally discovered as a conserved region responsible for the activation of PKCs by diacylglycerol or phorbol esters, C1 domains have been found in >200 other proteins. The C1 domain is a compact motif of ~50 amino acid residues, containing two small β sheets and short C-terminal α helix that are built around two 3-Cys-1-His Zn2+- binding clusters, with the two ions integral to the overall structure. One entire end of the C1 domain surrounding the diacylglycerol-binding groove is very hydrophobic. Membrane binding of C1 domains occurs by strong synergism between the stereospecific interaction of diacylglycerol with its binding site and the nonspecific hydrophobic interaction between the membrane and the C1 domain surface surrounding the binding site. In most PKCs, C1 domains occur in pairs. C1 domains from PKCγ have been observed to translocate from the cytosol to the plasma membrane within a few seconds after addition of diacylglycerol. In the inactive cytosolic form of PKCγ, the diacylglycerol- binding sites are obstructed. Opening of these sites may require binding of the enzyme to the membrane via Ca2+-mediated C2 domain-phospholipids interactions. Diacylglycerol binding to the C1 domain is also believed to lead to allosteric activation of the enzyme by a conformational change that alters the interactions of the C1 domains with the catalytic domain of the enzyme. 37
  38. 38. C2 domains C2 domains consist of ~120 residues folded in a β sandwich structure related to that of immunoglobulin. Originally discovered as a conserved motif in Ca2+-dependent PKCs, ~600 C2 domains have now been found in >400 proteins involved not only in signal transduction, but also in inflammation, synaptic vesicle trafficking and fusion, and many other processes. The properties of C2 domains vary, with some of them binding to phospholipid membranes in a Ca2+-dependent manner, while others bind constitutively. Other C2 domains exhibit both Ca2+-dependent and -independent binding to proteins rather that membranes. The structures of C2 domains from synaptotagmin, PKC-β and -δ, and phospholipases A2 (cPLA2) and C-δ1 (PLCδ1) have been determined. The Ca2+-binding sites are formed by three loops at one tip of the structure. Most Ca2+-dependent C2 domains bind acidic phospholipids, but that of cPLA2 seems to prefer neutral lipids, especially phosphatidylcholine (PC). The subcellular localization of C2 domains correlates with their phospholipid specificity. Thus, when the free Ca2+ concentration increases in response to a stimulus, C2 domains from PKCα and PKCγ translocate to the plasma membrane, rich in the acidic phosphatidylserine lipid, whereas cPLA2 translocates to the PC-rich nuclear envelope and endoplasmic reticulum. FYVE domains FYVE domains have been found in ~60 proteins and consist of 70-80 residues containing 8 Cys or 7 Cys and 1 His that coordinate two Zn2+. FYVE domains are specific for phosphatidylinositol-3-phosphate (PI3P), whose concentration in the cell increases following activation of phosphatidylinositol 3-kinases (PI3-kinase). As illustrated in the figure above, FYVE domains bind to PIP3-containing membranes so that the tip of the N-terminal loop, which contains hydrophobic residues, penetrates into the bilayer. Proteins containing FYVE domains localize to endosomal membranes containing PI3P, and this localization is blocked by inhibition of PI3-kinase. 38
  39. 39. PH domains Found in >500 proteins, PH domains bind various phosphorylated phosphatidyl- inositols (phosphoinositides) with different affinities and thus respond sensitively to the activities of phosphatidylinositol kinases, phosphatases and phospholipases. Of particular interest, signaling through PI3-kinases depends on PH domain-containing effectors, in addition to those containing FYVE domains. Structures are known for PH domain of several proteins, among which spectrin, PLCδ1, β-adrenergic receptor kinase (βARK) and insulin receptor substrate 1 (IRS-1). The PH domain structure contains two orthogonal antiparallel β sheets of three and four β strands, followed by a C-terminal α helix. The β sheets fold into a barrel-like structure, one end of which is capped by the α helix. The loops connecting the β strands are involved in ligand binding and vary substantially in sequence and structure between PH domains. Based on the binding to different phosphoinosite polyphosphates and inositol polyphosphates, PH domains have been classified into four groups. Group1 contains proteins such as Bruton’s tyrosine kinase (Btk), whose PH domains bind phosphatidylinositol 3,4,5-trisphosphate, PI(3,4,5)P3 with high specificity. Group 2 includes proteins such as PLCδ1 and βARK, whose PH domains have high affinity for PI(4,5)P2 and PI(3,4,5)P3 in vitro. In vivo, preferential binding to PI(4,5)P2 may occur as a consequence of the higher abundance of this lipid rather than discrimination against the 3-phosphorylated PI. Group 3 includes proteins such as Akt, also known as protein kinase B (PKB), whose PH domains bind preferentially PI(3,4)P2 and PI(3,4,5)P3. Group 4 is a heterogeneous group that includes proteins with relatively low affinity for all ligands mentioned above. The PH domain of PLC-γ binds 3-phosphoinositides, including PI3P, while the PH domains of PLCβ1 and PLCβ2 bind nonspecifically and with low affinity to neutral and acidic phospholipids. In addition to binding to membrane-bound phosphoinositides, PH domains display variable affinity for soluble inositol phosphates. For examples, the PH domain of PLCδ1 binds to PI(4,5)P2 in vesicles with micromolar affinity and to the soluble Ins(1,4,5)P3 with Kd = 210 nM. The higher affinity for the latter may be important in 39
  40. 40. product inhibition of the enzyme. Stimulation of PLC causes repartitioning of a fusion protein consisting of green fluorescent protein and PLCδ1 PH domain from the plasma membrane to the cytosol concomitant with the hydrolysis of PI(4,5)P2 in the membrane and formation of soluble Ins(1,4,5)P3. PH domains that bind 3-phosphorylated phosphoinositides, including those of Btk and Akt, have similarly been observed to translocate to the plasma membrane upon activation of PI3-kinases. Many of the interactions described for these membrane-targeting domains are of relatively low affinity and thus their physiological importance may be questioned. However, many important interactions appear to be weak “by design”, so that membrane binding of certain proteins may require the simultaneous presence of more than one ligand, the coincident activation of more than one signaling pathway. This is exemplified by PKCγ, which contains both diacylglycerol-binding C1 and Ca2+-phospholipid-binding C2 domains, and requires both the production of diacylglycerol and an increase in the concentration of free Ca2+ for full activation. 40
  41. 41. Intracellular Ligand-Gated Channels Regulation of ion channel activity can modulate many physiological processes, such as electrical excitability, secretion, and salt transport across epithelia. Most channel proteins are post translationally modified (e.g. through phosphorylation or through interactions with intracellular signaling molecules) but certain channels depend on such interactions in order to be gated open or closed. Here we will consider three examples of intracellular ligand-gated channels: a K channels that is inhibited by the ligand ATP, a K channel that is activated by G proteins and a non-selective cationic channel that is activated by cyclic nucleotides. ATP-sensitive K channels: This inwardly rectifying K channel is inhibited by cytosolic adenosine triphosphate (ATP), thus coupling the metabolic state of the cell to membrane electrical events. These channels are found in all types of muscle cells (skeletal, cardiac and smooth), in neurons, in renal tubular cells and in the β cells of the pancreas, where their physiologic role is best understood. In pancreatic β cells they regulate insulin secretion in response to glucose. These channels are normally active at rest and β cells are thus kept at negative resting potentials. After a meal, when glucose is metabolized and ATP is produced (actually the channel senses the increased ATP/ADP ratio) the channel is inhibited the cell depolarizes, fires action potentials, allowing entry of Ca and secretion of insulin. These channels are associated with a member of the larger ATP-binding cassette proteins, called the sulphonylurea receptor or SUR (other members of these transmembrane proteins include: P-glycoprotein or multidrug resistance protein, the cystic fibrosis transmembrane regulator or CFTR, etc.- see lecture 12) (Fig. 1). Figure 1. The inward rectifier Kir6.2 combines with the sulfonylurea receptor (SUR) to generate ATP- sensitive K currents. The association of the SUR and the K channel produces a functional channel that is blocked by sulphonylureas, drugs that constitute the principal treatment for adult onset diabetes. Similarly, the SUR association causes the K channel to be activated by SUR- binding drugs called potassium “channel openers” (e.g. diazoxide, pinacidil), and by nucleoside diphosphates (e.g. ADP) that bind SURs at the nucleotide binding folds (NBFs – Fig. 1). It has been established that there is an inverse relationship between ATP sensitivity and PIP2 levels and that the channel activity depends on the presence of PIP2. However, the details of the mechanism by which ATP inhibition or SUR-channel 41
  42. 42. interactions relate to channel gating by PIP2 are unclear but are the subject of an active area of research. Moreover, the physiological importance of regulation of PIP2 levels on the activity of this K channel is not clear. It is long known for example that at glucose concentrations below the threshold for stimulation of insulin secretion and electrical activity, muscarinic stimulation that leads to PLC activation initiates electrical activity and insulin release. Whether, this effect is mediated by reduction of PIP2 levels and enhanced sensitivity to ATP inhibition of the K channels remains to be shown. G protein-gated K channels: Using the cell-attached mode of the patch clamp technique it can be shown that extracellular application of ACh is effective in stimulating channel activity in an atrial patch, when perfused through the pipette but not through the bath (Fig 2). In their isolated patch, external signaling was limited to ACh in the patch pipette (since the membrane patch seems to be physically isolated at the sites of the gigaseal between the glass electrode and the plasma membrane from substances in the bath and from membrane molecules other than those within the patch) whereas internally, soluble second messengers (e.g. GTP) do have access to the cytoplasmic surface of the patch. This result has been used as evidence for the membrane delimited nature of the action of ACh. Using the whole-cell mode of the patch-clamp technique, it has been shown that the ACh effect proceeded via a pertussis toxin (PTX) sensitive G protein and that non-hydrolyzable GTP analogs could bypass signaling through the receptor and cause persistent stimulation of channel activity. Experiments with inside-out patches provided further evidence for the membrane delimited nature of the ACh signaling and the involvement of G proteins. Perfusion of inside-out patches with purified Gβγ subunits caused persistent stimulation Figure 2. Unitary inward K currents measured on a rabbit atrial cell with an on-cell patch pipette containing isotonic KCl. The control trace is before ACh additions. The second trace is after perfusion of 100 nM ACh in the bath, and the third trace is after washing ACh out of the bath and perfusing 10 nM ACh into the pipette. Em = -90 mV. 42
  43. 43. of K current activity in a Mg-independent manner. The Gβγ activation of this K current provided the first example of the effector function of the Gβγ complex in any system. It has been shown that Gβγ binds directly to both the carboxy- and amino- cytoplasmic segments of the channel protein. It is thought that Gβγ binding stabilizes channel-PIP2 interactions that serve to open the channel gate. The details of this mechanism are an active area of research. The atrial (or nodal) K+ channel that is activated by acetylcholine through muscarinic m2 type receptors has served as the prototypical G protein-gated K channel. Pertussis toxin-sensitive, neurotransmitter-activated inwardly rectifying K+ currents have also been reported in the central nervous system and in other peripheral tissues such as the pancreas and the pituitary. Direct activation of potassium (K+) channels by G proteins is involved in the rapid inhibition of membrane excitability, such as in the slowing of heart rate by the vagus nerve or the autoinhibitory release of dopamine by midbrain neurons. Thus these K channels couple G protein-coupled receptor signaling to membrane excitability. Figure 3. In the hypothesis of membrane-delimited signaling, drawn here for muscarinic modulation of a K(ACh) channel, only three macromolecules are used in the signaling cascade: receptor (M), G protein (Gk), and channel. They remain in the membrane throughout. The activated G protein (G*) interacts directly Figure 4 shows the crystal structure of the cytoplasmic domains of one of the G protein sensitive channels GIRK1. On this structure we have mapped mutations that affect Figure 4. Crystal structure of the cytosolic domains of GIRK1. Mutation sites have been mapped onto the structure revealing that PIP2 and Gβγ interacting sites of the channel are in close proximity to the lipid (Nishida and MacKinnon, 12/27 2002, Cell 111:958-965) 43
  44. 44. channel-Gβγ and channel-PIP2 interactions. Most of these mutations seem to come together to a region that is in close proxility to the lipid bilayer. Cyclic nucleotide-gated channels involved in phototransduction: Cyclic nucleotide-gated channels (CNG) are composed of two subunits in a tetrameric arrangement, two α subunits and two β subunits. The α subunits can produce functional channels when expressed in heterologous expression systems. The β subunits do not express by themselves, but when co-expressed with their corresponding α subunit, they produce channels with altered permeation, pharmacology, and/or cyclic nucleotide selectivity. The primary structure of each CNG channel subunit is a six-membrane spanning segment protein resembling that of voltage-gated K channels (we will examine those in the next lecture). CNG channels are found in all sensory organs and possess a cyclic nucleotide-binding domain. This is a highly conserved stretch of approximately 120 amino acids that is homologous to similar domains of other proteins, including the cAMP- and cGMP-dependent protein kinases and the catabolite-activating protein (CAP), a bacterial transcription factor. Although the retinal and olfactory CNG channels exhibit a high degree of sequence similarity (over 80% amino acid identity) in the putative binding region, the native channels exhibit different cyclic nucleotide selectivities. For the retinal channel, cGMP is a much more potent and effective agonist than cAMP. For the native olfactory channel cAMP and cGMP have very similar effects. Let us consider the role of retinal CNG channels in phototransduction. In the Figure 5. The retina has five major classes of neurons arranged into three nublear layers: photoreceptors (rods and cones), bipolar cells, horizontal cells, amacrine cells, and ganglion cells. Photoreceptors, bipolar, and horizontal cells make synaptic connections with each other in the outer plexiform layer. The bipolar, amacrine, and ganglion cells make contact in the inner plexiform layer. Bipolar cells bridge the two layers. Information flows vertically from photoreceptors to bipolar cells to ganglion cells. Information also flows laterally, mediated by horizontal cells in the outer plexiform layer and amacrine cells in the inner plexiform layer. retina of vertebrates phototransduction is accomplished by sensory cells (the rods and the cones), connected by interneurons (bipolar, horizontal and amacrine cells) to ganglion cells that transmit signals to the optic nerve and the brain (Fig. 5). Rods and cones have different sensitivities and respond to different frequencies of light. The 44
  45. 45. cones cells can further be subdivided into cells that preferentially sense different colors. However, it is rods, from a variety of species, that have been the favored cell type for studying visual transduction. Figure 6a shows the structure of a rod photoreceptor. Figure 6a. A photoreceptor. The drawing (left) is of an entire rod photoreceptor. The micrograph Figure 6b. The dark current (left). In the dark, (right) shows only the outer segment current flows through sodium channels in the of a salamander cone. outer segment of a rod photoreceptor. A pulse of light (right) closes these channels, resulting in hyperpolarization of the rod. The cell has two parts. The rod outer segment is elongated and contains a stack of flattened disks made from internal membranes. This is connected by a thin bridge to the remainder of the cell, the inner segment that contains the nucleus, the mitochondria, and the presynaptic terminal that synapses onto other neurons in the retina. It is the outer segment that is the business end for visual transduction. Within the internal membranous disks is found the light-sensitive protein rhodopsin. This is made up of an opsin protein, bound to a light-sensitive molecule or chromophore termed retinal. The later molecule may exist in a number of different forms, of which 11-cis-retinal and all- trans-retinal are the two major isomers. On its own, neither opsin nor retinal absorbs visible light. In combination, however, absorption of a photon of light causes and isomerization of retinal from the 11-cis form to the all-trans form. The light-dependent 45
  46. 46. isomerization of retinal then causes a structural rearrangement of the protein. Rhodopsin that has been activated in this way is termed meta-rhodopsin. For all of the subsequent steps in visual transduction, it is useful to think of this molecule as analogous to a receptor that has just bound its neurotransmitter. In fact, the structure of the opsin protein is similar to G-protein coupled receptors. Not surprisingly, therefore, the steps that follow the production of meta-rhodopsin involve the production of a second messenger through the action of a G-protein. This G-protein is called transducin. When meta-rhodopsin binds to transducin, GDP is replaced by GTP, and the αT-subunit of transducin is liberated from its complex with the βγ subunit. The target of the newly liberated αT is an enzyme in the membranous disks, a phosphodiesterase, that cleaves the second messenger cGMP to 5 ' GMP. Even in the dark, the levels of cGMP in the outer segments are maintained by a balance between its rate of synthesis through guanylate cyclase and degradation by the phosphodiesterase. The action of αT, formed after exposure to light, is to stimulate the phosphodiesterase, producing a drop in the levels of cGMP. This drop occurs within about 100 ms of the onset of a light flash, sufficiently fast to account for a visual response. In many respects, photoreceptors are built backwards. When excited by light, they respond by dropping, rather than raising, their concentration of the second messenger cGMP. The dominant type of ion channel in the plasma membrane of the outer segments is the CNG channel that allows sodium and calcium to enter the cell. Because of the abundance of sodium ions in the extracellular fluid, the major ion that enters the outer segments through these channels is sodium. We would expect a cell with a preponderance of such CNG channels to have a very positive resting potential. The effect of the rod CNG channels is, however, counterbalanced by potassium channels. The interesting thing about these potassium channels is that they are found in a very different part of the cell, the membrane of the inner segment that includes the nucleus and synaptic terminal. Because there is good electrical continuity between the inner and outer segments, the mean membrane potential is kept fairly negative as a result of the open potassium channels. This spatial distribution of channels, however, creates a circulating current, termed the dark current (Fig 6b), which flows in through the outer segment CNG channels, through the bridge into the inner segment and out through the potassium channels. The effect of shining light on a rod is to shut down many of the CNG channels in the outer segment. This produces a marked decrease in the dark current. As a result the potassium channels, which remain open in the inner segment, hyperpolarize the cell toward EK, reducing the spontaneous release of neurotransmitter from the synaptic terminal. The closure of the CNG channels can be attributed directly to the drop in cGMP in the cytoplasm of the outer segment. The CNG channels normally bind cGMP directly, and remain open only when cGMP is bound. This can be demonstrated by making inside-out patch recording on membrane from the outer segments. When cGMP is added to the cytoplasmic face of the patch a large increase in conductance, attributable to the opening of the CNG channels can be measured. One interesting feature of the CNG channels is that, under normal conditions, the conductance of a single channel is extremely low. The reason for this is that calcium and magnesium ions, which are normally present in physiological solutions, partially block these channels. This block is relieved when calcium and magnesium are omitted from the solutions, and individual openings of the channel are much larger. 46
  47. 47. This cascade of reactions that follows the formation of meta-rhodopsin produces a very significant amplification of the signal generated by light. It has been estimated that a single molecule of meta-rhodopsin, which is formed by the action of a single photon of light, diffuses in the membrane and activates several hundred transducin molecules before it is rendered inactive. The subsequent stimulation of the phosphodiesterase by αT provides further amplification such that a single photon of light can lead to the destruction of more than 100,000 molecules of cGMP. The analogies between visual transduction and neurotransmitter action can be taken further, when one considers how the response to a flash of light is terminated. A protein called rhodopsin kinase phosphorylates meta-rhodopsin making it relatively ineffective at activating transducin, and thus terminating the light response. After phosphorylation, the all-trans-retinal dissociates from rhodopsin, leaving the opsin protein, which must bind another 11-cis-retinal before it can again be activated by light. Similarly, the β-adrenergic receptor (stimulated by isoproterenol or norepinephrine) is phosphorylated by the β-adrenergic receptor kinase (βARK) to make it ineffective in stimulating Gs. Neurotransmitter Receptors The diversity of neurotransmitters is extensive, but their receptors can be grouped into two broad classes: ligand-gated ion channels and G protein-coupled receptors. In this section, we describe two important receptors that are also ligand-gated ion channels. By far the most- studied receptor is the muscle nicotinic acetylcholine receptor, the first ligand-gated ion channel to be purified, cloned, and characterized at the molecular level. The structure and mechanism of this receptor are understood in considerable detail, and it provides a paradigm for other neurotransmitter-gated ion channels. When activated, these receptors induce rapid changes, within a few milliseconds, in the permeability and potential of the postsynaptic membrane. In contrast, the postsynaptic responses triggered by activation of G protein-coupled receptors occur much more slowly, over seconds or minutes, because these receptors regulate opening and closing of ion channels indirectly. Opening of Acetylcholine-Gated Cation Channels Leads to Muscle Contraction The nicotinic acetylcholine receptor, a ligand-gated cation channel, admits both K+ and Na+. Although found in some neurons, this receptor is best known for its role in synapses between motor neurons and skeletal muscle cells. Patch-clamping studies on isolated outside-out patches of muscle plasma membranes have shown that acetylcholine causes opening of a cation channel in the receptor capable of transmitting 15,000-30,000 Na+ or K+ ions a millisecond. Since the resting potential of the muscle plasma membrane is near Ek, the potassium equilibrium potential, opening of acetylcholine receptor channels causes little increase in the efflux of K+ ions; Na+ ions, on the other hand, flow into the muscle cell. The simultaneous increase in permeability to Na+ and K+ ions produces a net depolarization to about –15mV from the muscle resting potential of –85 to –90 mV. This depolarization of the muscle membrane generates an action potential, which – like an action potential in a neuron – is conducted along the membrane surface via voltage-gated Na+ channels. When the membrane depolarization 47
  48. 48. reaches a specialized region, it triggers Ca2+ movement from its intracellular store, the sarcoplasmic reticulum, into the cytosol; the resultant rise in cytosolic Ca 2+ induces muscle contraction. Two factors greatly assisted in the characterization of the nicotinic acetylcholine receptor. First, this receptor can be rather easily purified from the electric organs of electric eels and electric rays; these organs are derived from stacks of muscle cells (minus the contractile proteins) and thus are richly endowed with this receptor. (In contrast, this receptor constitutes a minute fraction of the total membrane protein in most nerve and muscle tissues). Second, α- bungarotoxin, a neurotoxin present in snake venom, binds specifically and irreversibly to nicotinic acetylcholine receptors. This toxin can be used in purifying the receptor by affinity chromatography and in localizing it. For instance, in autoradiographs of muscle-cell sections exposed to radioactive α-bungarotoxin, the toxin is localized in the plasma membrane of postsynaptic striated muscle cells immediately adjacent to the terminals of presynaptic neurons. Careful monitoring of the membrane potential of the muscle membrane at a synapse with a cholinergic motor neuron has demonstrated spontaneous, intermittent, and random ~2-ms depolarizations for about 0.5-1.0 mV in the absence of stimulation of the motor neuron. Each of these depolarizations is caused by the spontaneous release of acetylcholine from a single synaptic vesicle. Indeed, demonstration of such spontaneous small depolarizations led to the notion of the quantal release of acetylcholine (later applied to other neurotransmitters) and thereby led to the hypothesis of vesicle exocytosis at synapses. The release of one acetylcholine-containing synaptic vesicle results in the opening of about 3000 ion channels in the postsynaptic membrane, far short of the number need to reach the threshold depolarization that induces an action potential. Clearly, stimulation of muscle contraction by a motor neuron requires the nearly simultaneous release of acetylcholine from numerous synaptic vesicles. All Five Subunit in the Nicotinic Acetylcholine Receptor Contribute to the Ion Channel 48
  49. 49. The acetylcholine receptor from skeletal muscle is a pentameric protein with a subunit composition of α2βγδ. Each molecule has a diameter of about 9nm and protrudes about 6nm into the extracellular space and about 2nm into the cytosol (Figure 11-36). The α, β, γ, and δ subunits have considerable sequence homology; on average, about 35-40 percent of the residues in any two subunits are similar. The complete receptor has a five-fold symmetry, and the actual cation channel is a tapered central pore, with a maximum diameter of 2.5 nm, formed by segments from each of the five subunits (Figure 11-36). The channel opens when the receptor cooperatively binds two acetylcholine molecules to sites located at the interfaces of the αδ and αγ subunits. Once acetylcholine is bound to a receptor, the channel is opened virtually instantaneously, probably within a few microseconds. Studies measuring the permeability of different small cations suggest that the open ion channels is, at its narrowest, about 0.65-0.80 nm in diameter, in agreement with estimates from electron micrographs. This would be sufficient to allow passage of both Na+ and K+ ions with their bound shell of water molecules. Although the structure of the central ion channel is not known in molecular detail, much evidence indicates that it is lined by five transmembrane M2 α helices, one from each of the five subunits. The M2 helices are composed largely of hydrophobic or uncharged polar amino acids, but negatively charged aspartate or glutamate residues are located at each end, near the membrane faces and several serine or threonine residues are near the middle. If a single negatively charged glutamate or aspartate in one subunit is mutated to a positively charged lysine, and the mutant mRNA is injected together with mRNAs for the other three wild-type subunits into frog oocytes, a functional channel is expressed, but its ion conductivity – the number of ions that can cross it during the open state – is reduced. The greater the number of glutamate or aspartate residues mutated (in one or multiple subunits), the greater that reduction in conductivity. These findings suggest that aspartate and glutamate residues – one residue 49
  50. 50. from each of the five chains – form a ring of negative charges on the external surface of the pore that help to screen out anions and attract Na+ or K+ ions as they enter the channel (see Figure 11-36). A similar ring of negative charges lining the cytosolic pore surface also helps select cations for passage. The two acetylcholine-binding sites in the extracellular domain of the receptor lie ~4 to 5 nm from the center of the pore. Binding of acetylcholine thus must trigger conformational changes in the receptor subunits that can cause channel opening at some distance from the binding sites. Receptors in isolated postsynaptic membranes can be trapped in the open or closed state by rapid freezing in liquid nitrogen. Images of such preparations suggests that the five M2 helices rotate relative to the vertical axis of the channel during opening and closing. Two Types of Glutamate-Gated Cation Channels May Function in a Type of “Cellular Memory” The hippocampus is the region of the mammalian brain associated with many types of short- term memory. Certain types of hippocampal neurons, here simply called postsynaptic cells, receive inputs from hundreds of presynaptic cells. In long-term potentiation a burst of stimulation of a post-synaptic neuron makes it more responsive to subsequent stimulation by presynaptic neurons. For example, stimulation of a hippocampal presynaptic nerve with 100 depolarizations acting over only 200 milliseconds causes an increased sensitivity of the postsynaptic neuron that lasts hours to days. Changes in the responses of postsynaptic cells may underlie certain types of memory. 50
  51. 51. Two types of glutamate-gated cation channels in the postsynaptic neuron participate in long- term potentiation. Unlike other neurotranmitter-gated ion channels, both glutamate receptors have four subunits, each containing a pore-lining M2 helix; both are excitatory receptors, causing depolarization of the plasma membrane when activated. Because the two receptors were initially distinguished by their ability to be activated by the non-natural amino acid N- methyl-D-aspartate (NMDA), they are called NMDA glutamate receptors and non-NMDA glutamate receptors. As illustrated in Figure 11-41, non-NMDA receptors are “conventional” in that binding of glutamate, released from the presynaptic cell, triggers their opening. NMDA glutamate receptors are different in two key respects. First, they allow influx of Ca 2+ as well as Na+. Second, and more important, two conditions must be fulfilled for the ion channel to open: glutamate must be bound and the membrane must be partly depolarized. In this way, the NMDA receptor functions as a coincidence detector; that is, it integrates activity of the postsynaptic cell – reflected in its depolarized plasma membrane – with release of neurotransmitter from the presynaptic cell, generating a cellular response greater than that caused by glutamate release alone. Once a post-synaptic cell becomes “sensitized”, it takes fewer action potentials in the presynaptic neurons to induce a given depolarization in the postsynaptic neuron; in other words, the synapse “learns” to have an enhanced response to signals from the presynaptic cells. 51
  52. 52. Opening of NMDA receptors depends on membrane depolarization because of the voltage- sensitive blocking of the ion channel by a Mg2+ ion from the extracellular solution. A small depolarization of the membrane causes the Mg 2+ ion to dissociate from the receptor, thereby making it possible for glutamate binding to open the channel. Mutagenesis of a single asparagine residue in the pore-lining M2 helix of the NMDA receptor abolishes the effect of Mg2+, indicating that Mg2+ binds in the channel. Since activation of a single synapse, even at high frequency, generally causes only a small depolarization of the membrane of the postsynaptic cell, long-term potentiation is induced only when many synapses simultaneously stimulate a single postsynaptic neuron. Thus, the requirements for membrane depolarization explains why long-term potentiation depends on the simultaneous activation of a large number of synapses on the postsynaptic cell. 52