Ionic Equilibria and Membrane Potential
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An introductory lecture the ionic equilibria and membrane potential.
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Ionic Equilibria and Membrane Potential
- 1. Ionic Equilibria & Membrane Potential Csilla Egri KIN 306 Spring 2012 Not everyone has to look so haggard doing science…
- 2. Outline Membrane potential Nernst and GHK equations Electrophysiology Methods Current measurements Voltage-gated ion channels Na channel K channel Channelopathies 2
- 3. Membrane potential - Review 3 Figure 7-1 Kandel electrochemical membrane potential (Vm) exists due to: a) differences in ion concentrations on opposite sides of the membrane b) selective permeability of membrane to various charged ions Neuronal resting Vm ≈ -60 to -70mV What molecule(s) are responsible for the excess intracellular negative charge?
- 4. Selective permeability 4 Figure 6-9 B&B Cell permeability to any one ion changes with opening/closing of ion channels Direction of movement of one ion dictated by the electrochemical driving force: (Vm - Eion) Membrane potential Nernst potential Driving force
- 5. Nernst Potential 5 3 equivalent definitions: Predicts Vm if membrane were permeable to only ion ‘x’ The membrane potential at which there is no driving force (no net influx/efflux) for ion ‘x’ = equilibrium potential The membrane potential above which the direction of flux of ion ‘x’ reverses = reversal potential RT/F = 60 mV at 310 K (37° C) in out X X X zF RT E ][ ][ ln= (Vm - Eion) Membrane potential Nernst potential Driving force
- 6. Goldman-Hodgkin-Katz equation6 Predicts actual resting membrane potential (Vm) Accounts for membrane permeability (conductance) to all major ions oCliNaiK iCloNaoK m ClPNaGKG ClGNaGKG zF RT v ][][][ ][][][ ln −++ −++ ++ ++ = Due to non-voltage gated K+ “leak” channels, at rest: GK>>GNa>GCl Therefore resting Vm approaches that of the most permeant ion (E )
- 7. Typical ionic concentration gradients7 Ion Extracellu lar (mM) Intracellu lar (mM) Enernst (mV at 37ºC) Na+ 145 12 +67 K+ 4.5 155 -95 Ca2+ 1 10-4 +123 Cl- 116 4.2 -89 What would happen to Vm if extracellular [K+] increased (hyperkalemia)? What if extracellular [K+] decreased (hypokalemia)?
- 8. Maintenance of ionic concentration gradients8 Na/K pump: uses energy from ATP to transport 3Na+ out for every 2K+ in Electrogenic pump (creates a net movement of positive charge out of the cell movement of charged ions = current) Na/Ca exchanger: Uses electrochemical energy of Na+ to drive efflux of Ca2+ (3Na+ in for one Ca2+ out) Electrogenic Cation-chloride cotransporters: Use electrochemical gradients of cations to transport chloride into or out of cell (depends on type of transporter) Electroneutral
- 9. Total membrane current (Im) 9 Total membrane current (Im) is the sum of two components: Im = Ii + Ic Ionic current (Ii) movement of ions across the membrane through channels Ii = Gion x (Vm - Eion) Ionic current Capacitative current Ionic conductanc e Membrane potential Nernst potential Driving force
- 10. Membrane capacitance (Cm) 10 Figure 6-9 B&B Membrane capacitance (Cm) determines the ability to separate charges of opposite sign The charge (Q) stored by a capacitor is the product of capacitance and voltage d A Cm κ = mmVCQ =
- 11. Capacitative current (IC) 11 capacitive current (Ic) equal to the rate of change in charge separation does not require movement of ions across the cell membrane, just change in Vm Always occurs when the membrane experiences a change in voltage. Always has an exponential decay Does not tell us anything about ion channel function Can be used to indicate relative size of the cell tVCtQI mmc // ∆=∆= Fig 6-12 B&B Ic Ic d A Cm κ = mmVCQ =
- 12. Electrophysiology – current clamp12 Electrodes inserted in the cell membrane record the difference in membrane potential between the inside and outside of the cell Current clamp injects current and observes resulting voltage changes Fig 6-12 B&B
- 13. Electrophysiology – voltage clamp13 Fig 6-13 B&B voltage clamp holds membrane voltage constant and observes resulting current changes Two common methods: Two-electrode voltage clamp for large cells (squid giant axon, xenopus laevis oocytes) Electrodes actually impale cell Patch clamp for smaller cells (mammalian cells, cultured neurons) Glass pipette filled with electrolyte solution makes contact with cell membrane Ic IcIi
- 14. Electrophysiology – voltage clamp14 Two-electrode voltage clamp Patch clamp Fig 6-13,14 B&B
- 15. Ionic Current Measurements 15 Figure 6-13 B&B Typical ionic current trace in response to a depolarizing stimulus when a cell membrane contains voltage gated Na+ Ii Ii
- 16. Voltage Gated Na+ Channel (NaV) Structure Figure 7-12 B&B Inactivation gate Inactivation gate 4 homologous domains (D1-D4) each with 6 transmembrane spanning segments (S1-S6) Tertiary protein structure folds to form central aqueous pore (the α subunit, pore lined by S5-S6) Accessory β subunits modulate channel gating and trafficking to the membrane 9 isoforms (NaV1.1-1.9) localized to different tissues 16
- 17. Voltage Gated Na+ Channel (NaV) Structure Figure 7-12 B&B Inactivation gate Inactivation gate 17 voltage sensors – positively charged S4 segments. Move across electric field (membrane) in response to changes in Vm. Movement of all four S4s leads to channel activation. activation gate - in center of channel pore normally closed at resting membrane potential inactivation gate – intracellular linker between D3 and D4 normally open at resting membrane potential occludes channel pore (closes) shortly after activation time and voltage dependent
- 18. Voltage Gated Na+ Channel (NaV) Function18 Determines the rising phase of the action potential Probability of activation gate opening increases with increasing membrane depolarization. at apprx -55mV, enough NaV channels open to initiate all-or-none action potential inactivation gate closes about 1-2 ms later NaV channels cannot be reactivated (opened) until inactivation gate re-opens (near resting Vm)
- 19. Voltage Gated Na+ Channel (NaV) Structure related to function19 Membrane potential(mV)Ioniccurrent(nA) Time (ms) 0 15 -70 +10 0 -2 1 2 3 4 5 1 2 3 4 5
- 20. Voltage gated K+ Channels (Kv) Structure20 Figure 7-12 B&B 4 α subunits each with 6 transmembrane segments (S1-S6) Tertiary protein results from assembly of the 4 α subunits creating a central aqueous pore lined by S5-S6 One accessory β subunit modulates channel function 40 isoforms with different gating and current generating properties localized to different tissues
- 21. Voltage gated K+ Channels (Kv) Function21 Have two important divisions, Kv channels that mediate either: Delayed outward rectifying currents Activation has a sigmoidal, delayed lag phase Responsible for the downward phase of the action potential Transient A-type outward rectifying currents Activate and inactivate over a short time scale Important in determining interval between action potentials Both pass only outward current B&B pg. 197
- 22. Voltage gated K+ Channels (Kv) Function22 Gao B , Ziskind-Conhaim L J Neurophysiol 1998;80:3047-3061 Control: current trace depicting all types of Kv currents in a mouse motorneuron IK: non-inactivating delayed rectifier K current IA: transient A-type K current
- 23. Voltage dependence of activation23 Figure 7-7B&B Na+ K+ When P0=0.5 half the channels are open, half are closed. The Vm that this occurs at is called the V1/2 of activation
- 24. Macroscopic current-voltage relationships24 Where on this figure is the equilibrium potential for K+ and Na+ ? Why is the I-V relationship for Na+ biphasic? Figure 7-6 B&B Ii = Gion x (Vm - Eion)
- 25. Channelopathies 25 Amino acid mutations in ion channels leading to improper protein function and disease Schematic of NaV1.4 and associated disease causing mutations Figure 7-15 B&B
- 26. Paramyotonia congenita (PMC) 26 PMC patients have no symptoms at warm temperatures, but are subject to cold induced myotonia (muscle stiffness). With intensive cooling, the myotonia can give way to periodic paralysis of the muscles. Caused by mutations to NaV1.4, predominant in skeletal muscle, that impair inactivation of the channel Mild impairment to inactivation: results in small depolarizing current, bringing the membrane slightly closer to threshold and increasing cellular excitability myotonia or stiffness Severe impairments to inactivation: can significantly depolarize membrane (from -90mV to -40mV) placing unmutated channels in the inactivated state membrane is refractory muscle weakness or paralysis Both genotype and phenotype are heterogeneous Why cold exacerbates PMC symptoms is yet to be determined. WebCT readings: Paramyotonia Congenita
- 27. Objectives After this lecture you should be able to: Describe what gives rise to the membrane potential and how ionic concentration gradients are maintained Describe the ways in which electrical properties of membranes can be measured and distinguish between current clamp and voltage clamp Define the components of total membrane current List the key features of voltage gated sodium and potassium channels, including structure, voltage-dependence of activation and current-voltage relationships Explain the causes of PMC and distinguish between the molecular causes of myotonia and weakness or paralysis 27
- 28. 28 1. Would you use voltage or current clamp to observe action potentials in neurons in response to excitatory neurotransmitters? 2. At a membrane potential of -30mV, would there be more Na+ or K+ current? 3. If a person with PMC had elevated serum K+ levels, would this increase or decrease the likelihood of experiencing an episode of myotonia? Test your knowledge
Editor's Notes
- Readings B&B Chapter 3, pages 50 - 69 Chapter 6, pages 145 - 163
- CH 7 and 6 Kandell
- You already learned a lot about channels and transporters in the renal and GI sections of the course. We are simply shifting our focus to neuronal cells now, which have a voltage gradient across their membranes. Organic anions, such as proteins and amino acids (385mM intracellular) Since opposite charges attract each other, the excess negative and positive charges collect locally on the intra and extra cellular faces The electrochemical gradient, or electrochemical potential difference is used to quantitate the driving force acting on a molecule to cause it to move across a membrane. It is a measure of the free energy available to carry out the useful work of transporting the molecule across the membrane. It has two components. One represents the energy in the concentration gradient (the chemical potential difference) and the second represents the energy associated with moving charged particles (the electrical potential difference).
- At rest, a nerve cell is almost exclusively permeable to K via “leak” channels. The electrochemical driving force dictates the movement of that ion. Next we will look at how to describe the driving force for movement of ions
- Since R (gas constant) and F (Faraday constant) are constants, RT/zF can be calculated for any temperature (T) or ion valence (z) Used to determine the equilibrium or reversal potential of a single ion (e.g. K+, Na+, Ca2+, or Cl-) In using the Nernst equation, it is often convenient to convert the equation to a form that involves logarithm to the base 10 (log) rather than natural logarithms (ln). The formula for this conversion is ln y = 2.303 log y. Because biological electrical potentials are usually expressed in millivolts (mV), the units of R may be selected so that RT/F comes out in millivolts. At 29.2° C, the quantity 2.303 RT/F is equal to 60 mV. Because this quantity is proportional to the absolute temperature, it changes by approximately 1/273 (0.36%) for each centigrade degree. Thus the Nernst Equation can also be expressed as -60 mV/z log [X+]in/[X+]out or 60 mV/z log [X+]out /[X+]in In brief, the Nernst equation can be used to predict the direction that ions tend to flow: If the potential difference measured across a membrane is equal to the potential difference calculated from the Nernst equation for a particular ion, that ion is in electrochemical equilibrium across the membrane, and no net flow of that ion will occur across the membrane. If the measured electrical potential is of the same sign (positive or negative) as that calculated from the Nernst equation for a particular ion but is larger in magnitude than the calculated value, the electrical force is larger than the concentration force. Therefore, net movement of that particular ion tends to occur in the direction determined by the electrical force. Figure 2-3 meets this condition. When the electrical potential difference is of the same sign but is numerically less than that calculated from the Nernst equation for a particular ion, the concentration force is larger than the electrical force. Therefore, net movement of that ion tends to occur in the direction determined by the concentration difference. If the sign of the electrical potential difference measured across the membrane is opposite to that predicted by the Nernst equation for a particular ion, the electrical and concentration forces are in the same direction. Thus, that ion cannot be in equilibrium, and it will tend to flow in the direction determined by both electrical and concentration forces.
- Start Jan 09
- Hyperkalemia = nernst potential for K would become more depolarized, less electrochemical driving force for K efflux, and since Vm is proportional to the ion that is most permeable at rest, Vm would also become more depolarized (hyperexcitable state) Hypokalemia = nernst potential for K would become more hyperpolarized, a greater electrochemical driving force for K efflux, Vm would become more negative. (Hypoexcitable state)
- Na+/K+ pump: counteracts movement of ions due to electrochemical potential and maintains concentration gradients 3 Na+ pumped out, 2 K+ pumped in energy supplied by ATP hydrolysis Na+/ K+ pump is electrogenic produces a net movement of charge thus has a small negative contribution on the resting membrane potential
- Ionic current is directly proportional to the electrochemical driving force (Ohm’s Law: V=IR). Ionic current depends on the difference between actual Vm and Ex - it is proportional to the difference Vm - Ex, and the proportionality constant is the ionic conductance gx. Capacitive current is proportional to the rate of voltage change. I.e. capacitive current only occurs when Vm is changing. (Ionic current are the only current that flow across the membrane in the steady state, such as during an action potential, when Vm is constant.) The amount of charge accumulated along the membrane can occur more quickly than ionic current. In steady state (Vm constant) only ionic current flows across membrane. When membrane potential changes there is both ionic current and capacitive current. When membrane capacitance charges or discharges, the amount of stored charge changes so the membrane potential changes. If the circuit consists only of capacitor and resistor then membrane potential changes in exponential fashion {Fig. 6-11 B&B} The time constant = RC determines how fast V changes ( is time required for voltage to fall to 37% of its initial value V0). Capacitive current exists only when stored charge is changing.
- Electrical properties of model cell membranes. A, Four different ion channels are arranged in parallel in the cell membrane. B, The model represents each channel in A with a variable resistor. The model represents the Nernst potential for each ion as a battery. Notice that the four parallel current paths correspond to the four parallel channels in A. Also shown is the membrane capacitance, which is parallel with each of the channels. C, On the left is an idealized capacitor, which is formed by two parallel plates, each with an area, A, and separated by a distance, d. On the right is a capacitor that is formed by a piece of lipid membrane. The two plates are, in fact, the electrolyte solutions on either side of the membrane.
- Electrical properties of model cell membranes. A, Four different ion channels are arranged in parallel in the cell membrane. B, The model represents each channel in A with a variable resistor. The model represents the Nernst potential for each ion as a battery. Notice that the four parallel current paths correspond to the four parallel channels in A. Also shown is the membrane capacitance, which is parallel with each of the channels. C, On the left is an idealized capacitor, which is formed by two parallel plates, each with an area, A, and separated by a distance, d. On the right is a capacitor that is formed by a piece of lipid membrane. The two plates are, in fact, the electrolyte solutions on either side of the membrane.
- In panel "A" of Figure 6-12 in the textbook ("current clamp"), we instruct the electronics to suddenly increase the current that we are injecting into the cell, and to hold this new current at a constant value. The sudden increase in the current flowing through the membrane causes Vm to rise exponentially until we fully charge the membrane capacitance (Cm). Thus, Vm rises with a time constant (see webnote 0157a--Units for the 'Time Constant' (II5)) of Rm ´ Cm (Rm is membrane resistance). At infinite time, the charge on the capacitor is at its maximal value, and all the current flowing through the membrane flows through Rm, the "ohmic" membrane resis-tance. In panel "B" of Figure 6-12 in the textbook ("voltage clamp"), we instruct the electronics to inject enough current into the cell to suddenly increase in the membrane potential (Vm) of the cell. The current required to charge the membrane capacitance (Cm) is at first extremely large. However, as we charge the membrane capacitance, that current decays exponentially with a time constant (see webnote 0157a--Units for the 'Time Constant' (II5)) Rm ´ Cm. At infinite time, the membrane capacitance is fully charged, and no current is required to hold the command volt-age. However, this current decays exponentially, with a time course also determined by the R ´ C of the membrane.
- In panel "A" of Figure 6-12 in the textbook ("current clamp"), we instruct the electronics to suddenly increase the current that we are injecting into the cell, and to hold this new current at a constant value. The sudden increase in the current flowing through the membrane causes Vm to rise exponentially until we fully charge the membrane capacitance (Cm). Thus, Vm rises with a time constant (see webnote 0157a--Units for the 'Time Constant' (II5)) of Rm ´ Cm (Rm is membrane resistance). At infinite time, the charge on the capacitor is at its maximal value, and all the current flowing through the membrane flows through Rm, the "ohmic" membrane resis-tance. In panel "B" of Figure 6-12 in the textbook ("voltage clamp"), we instruct the electronics to inject enough current into the cell to suddenly increase in the membrane potential (Vm) of the cell. The current required to charge the membrane capacitance (Cm) is at first extremely large. However, as we charge the membrane capacitance, that current decays exponentially with a time constant (see webnote 0157a--Units for the 'Time Constant' (II5)) Rm ´ Cm. At infinite time, the membrane capacitance is fully charged, and no current is required to hold the command volt-age. However, this current decays exponentially, with a time course also determined by the R ´ C of the membrane.
- Two microelectrodes impale a Xenopus oocyte. One electrode monitors membrane potential (Vm) and the other passes enough current (Im) through the membrane to clamp Vm to a predetermined command voltage (VCommand). B, In the left panel, the membrane is clamped for 10 ms to a hyperpolarized potential (40 mV more negative). Because a hyperpolarization does not activate channels, no ionic currents flow. Only transient capacitative currents flow after the beginning and end of the pulse. In the right panel, the membrane is clamped for 10 ms to a depolarized potential (40 mV more positive). Because the depolarization opens voltage-gated Na+ channels, a large inward Na+ current flows, in addition to the transient capacitative current. Adding the transient capacitative currents in the left panel to the total current in the right panel, thereby canceling the transient capacitative currents (IC), yields the pure Na+ current shown at the bottom in the right panel.
- Downward spike discharging, upward spike charging of the membrane
- The voltage-gated Na+ channel has three known subunits: a large glycoprotien called the alpha-subunit, which probably forms the channel's pore, and two smaller polypeptides called β1 and β2 which regulate the function of the α (Kandel, 2000, p. 164). γ- and δ-subunits may also exist to regulate the α-subunit. The α-subunit has four repeats, labeled I through IV, of the same 150 amino acid sequence. Each repeat contains six membrane-spanning regions labeled S1 through S6 (Kandel, 2000, p. 164). The highly conserved S4 region, thought to be the part of the channel that acts as its voltage sensor, has a positive amino acid at every third spot, with hydrophobic residues between these (Kandel, 2000, p. 164). It is thought that when stimulated by a change in transmembrane voltage, this subunit moves from within the pore toward the extracellular side of the cell, allowing the channel to become permeable to ions which would otherwise have been blocked by the subunit's positive charges. The inner pore of sodium channels contains a selectivity filter made of negatively charged amino acid residues, which attract the positive Na+ ion and keep out negatively charged ions such as chloride (Kandel, 2000, p. 163). The cations flow into a more constricted part of the pore that is 0.3 by 0.5 nm wide, which is just large enough to allow a single Na+ ion with a water molecule associated to pass through (Kandel, 2000, p. 163-164). The larger K+ ion cannot fit through this area. Differently sized ions also cannot interact as well with the negatively charged glutamic acid residues that line the pore (Kandel, 2000, p. 163-164). (from Wikipedia: http://en.wikipedia.org/wiki/Sodium_ion_channel)
- The voltage-gated Na+ channel has three known subunits: a large glycoprotien called the alpha-subunit, which probably forms the channel's pore, and two smaller polypeptides called β1 and β2 which regulate the function of the α (Kandel, 2000, p. 164). γ- and δ-subunits may also exist to regulate the α-subunit. The α-subunit has four repeats, labeled I through IV, of the same 150 amino acid sequence. Each repeat contains six membrane-spanning regions labeled S1 through S6 (Kandel, 2000, p. 164). The highly conserved S4 region, thought to be the part of the channel that acts as its voltage sensor, has a positive amino acid at every third spot, with hydrophobic residues between these (Kandel, 2000, p. 164). It is thought that when stimulated by a change in transmembrane voltage, this subunit moves from within the pore toward the extracellular side of the cell, allowing the channel to become permeable to ions which would otherwise have been blocked by the subunit's positive charges. The inner pore of sodium channels contains a selectivity filter made of negatively charged amino acid residues, which attract the positive Na+ ion and keep out negatively charged ions such as chloride (Kandel, 2000, p. 163). The cations flow into a more constricted part of the pore that is 0.3 by 0.5 nm wide, which is just large enough to allow a single Na+ ion with a water molecule associated to pass through (Kandel, 2000, p. 163-164). The larger K+ ion cannot fit through this area. Differently sized ions also cannot interact as well with the negatively charged glutamic acid residues that line the pore (Kandel, 2000, p. 163-164). (from Wikipedia: http://en.wikipedia.org/wiki/Sodium_ion_channel)
- inactivation gate does not re-open (channels don’t recover from inactivation) until Vm returns toward resting value
- In cell biology, potassium channels are the most common type of ion channel. They form potassium-selective pores that span cell membranes. Potassium channels are found in most cells, and control the electrical excitability of the cell membrane. In neurons, they shape action potentials and set the resting membrane potential. They regulate cellular processes such as the secretion of hormones, so their malfunction can lead to diseases. Some potassium channels are voltage-gated ion channels that open or close in response to changes in the transmembrane voltage. They can also open in response to the presence of calcium ions or other signalling molecules. Others are constitutively open or possess high basal activation, such as the resting potassium channels that set the negative membrane potential of neurons. When open, they allow potassium ions to cross the membrane at a rate which is nearly as fast as their diffusion through bulk water. There are over 80 mammalian genes that encode potassium channel subunits. The pore-forming subunits of potassium channels have a homo- or heterotetrameric arrangement. Four subunits are arranged around a central pore. All potassium channel subunits have a distinctive pore-loop structure that lines the top of the pore and is responsible for potassium selectivity.
- Delayed outward rectifying: the activation of the current has a delayed, sigmoidal lag phase. Rectifying because outward current rises steeply at positive voltages (current flows only in outward direction). Transient A-type: also outwardly rectifying
- Large transient A-type K+ currents (I A) were elicited in embryonic motoneurons, and their amplitude did not increase after birth. A: total K+ currents (control, left) were generated in an E16 motoneuron by a series of depolarizing pulses to membrane potential of −30 to +50 mV (in 20-mV increments) applied from a HP of −80 mV. To isolate the transient K+ currents from the noninactivating delayed rectifier-type currents (I K), the latter were produced at the same depolarizing pulses that were preceded by a short (35 ms) prepulse to −40 mV (middle). At each depolarizing test potential, I A was obtained by subtracting I K from total K+ current (right). B: similar I A voltage dependence was apparent in E15–16 and P1–3 motoneurons. Threshold for I A activation was −30 mV. Values are means ± SE for n = 12–14.
- Maximum conductance for NaV channels occurs at about -25mV, for Kv channels about -10mV
- Here Ena is apprx. 50mV according to ionic concentrations. Ek is -80mV Voltage dependence of ionic currents. A, The top panels show the time course of the total ionic current. This is a voltage-clamp experiment on a frog node of Ranvier. Suddenly shifting Vm from a holding potential of -60 mV to -45, -30, 0, +30, and +60 mV elicits ionic currents that depend on Vm. B, These results are comparable to those in A, except that TEA abolished the outward K+ currents, leaving the Na+ current. Notice that the "peak" Na+ current varies with Vm. C, These results are comparable to those in A, except that TTX abolished the inward Na+ currents, leaving the K+ current. Notice that the "peak" K+ current varies with Vm. D, The blue curve is a plot of peak Na+ currents from experiments that are similar to those in B. The green curve is a plot of peak K+ currents from experiments that are similar to those in C. Notice that both the Na+ and the K+ currents are linear or "ohmic" in the positive voltage range. In a more negative Vm range, the Na+ current exhibits "negative resistance"; that is, the magnitude of the current becomes more negative rather than positive as Vm increases in the positive direction. TEA, tetraethylammonium; TTX, tetrodotoxin. A-C, data from Hille B: Common mode of action of three agents that decrease the transient change in sodium permeability in nerves.
- What is important between D3 and D
- PMC patients are asymptomatic at warm temperatures, but are subject to cold induced myotonia (muscle stiffness) aggravated by increased activity (paradoxical myotonia). With intensive cooling, the myotonia can give way to periodic paralysis of the muscles.24, 31 PMC is caused by mutations in the skeletal muscle sodium channel isoform NaV1.4. Most PMC mutations impair channel inactivation, leading to persistent INa and subsequent reduction of membrane excitability responsible for the periodic paralysis phenotype, while others increase membrane excitability resulting in muscle stiffness.26, 31, 32 This may seem counterintuitive, but a gradient exists between severity of inactivation impairment and resulting phenotype. Minor impairments to FI (between 8-20% non-inactivating channel population) result in a late INa that depolarizes the membrane just enough to bring it closer to AP threshold, thus increasing the probability of AP firing and muscle stiffness.33 Severe impairments to FI (greater than 20% non-inactivating channel population) however, result in a late INa that can significantly depolarize the membrane to about -40 mV (from a normal resting value of about -90 mV).33 At this potential, many remaining channels are in the inactivated state, rendering the membrane refractory and resulting in weakness or paralysis. Why cold or. in paradoxical myotonia, warmth exacerbate PMC symptoms is yet to be determined.