Short term paper on Mechanosensitive Channel of Small Conductance (MscS)

1. Mechanosensitive Channel of Small Conductance
(MscS)
BSE638 Term Paper
Chintalagiri Shashank, Y5157
May 9, 2010
1 Introduction and biological
significance
Mechanosentive channels act as floodgates in bacterial
cells, allowing them to respond to osmostic shock by
bulk removal of large amounts of water into the extra-
cellular area. In the event of osmotic downshock, for
instance, where the extracellular environment changes
suddenly to fresh water, the osmotic pressure differen-
tial across the cell membrane causes the cell to rapidly
uptake water. This, in turn, puts a lot of pressure
on the cell membrane which cannot be tolerated by
it. Mechanosenstive channels activate under such cir-
cumstances, and create a large channel for water to be
released from the cell.
Such proteins were first discovered about 20 years
ago, when Kung and co-workers identified stretch-
activated (mechanosensitive) proteins in bacterial
membranes that sense the increase in membrane ten-
sion during osmotic downshock. Two major families
of prokaryotic mechanosensitive channels were subse-
quently cloned: the mechanosensitive channel of large
conductance (MscL), and the mechanosensitive chan-
nel of small conductance (MscS). More recently, ad-
vances in techniques to crystallize and study membrane
proteins has allowed the open form structures of these
proteins to be trapped in crystals and studies, pro-
viding new insights into the mechanisms involved in
the process of maintaining the osmotic balance and the
functioning of these channels.
In particular, two very different approaches were
used to study MscS in its open form. In one approach,
a mutated version of the protein was used which was
trapped and crystallized in its ’open’ configuration af-
ter the application of high osmotic shock, since the
mutation was designed to stabilize the open state. In
the other approach, the native protein was trapped in
its open state using a lipid substrate that was intro-
duced after the pores were forced into the open state
by osmotic shock. SDSL based spectroscopic methods
were used to study the conformation of the molecule.
2 Structure of the Protein
MscS is a heptameric assembly, symmetric about the
axis normal to the membrane.
Figure 1: MscS structure [2]
The transisition between the open and closed form
is described as an iris like mechanism, similar to those
used in shutters in cameras, and involves the move-
ment of transmembrane α helices. While this is the
case with most ion channels in general, in the case of
MscS it takes place by reorientation of the correspond-
ing helices. In the wild type structure depicting the
closed state, it is seen that the transmembrane helices
(TM1,2,3a) are at an angle to to the normal to the
membrane. In the open state, however, the same he-
lices are seen to have rotated to be almost parallel to
the membrane normal. TM3b, however, shows little
movement.
Computational studies indicate that hydrophobic
pores of radii smaller than 4.5 or 6.5 ˚A are closed to
conduction of water and ions, respectively; these re-
sults imply that nonconducting pores need not be ge-
ometrically closed[1]. It is seen that the pores in both
mechanosensitive channel families are larger than this
in the open state (13 ˚A in MscS[2]) when in the ’open’
state, and are therefore quite capable of conducting
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2. Figure 2: MscS pore :This image shows the struc-
ture of the pore in the open(brown) and
closed(purple) states.[2]
Figure 3: MscS pore residues : This image shows the
structure of the pore in the open(brown)
and closed(purple) states, particularly the
Lyseine residues that block the pore when
closed are pulled apart by the iris like action
of TM3a[2]
water and ions. It has also been seen that in the closed
form, an intricate network of hydophobic side chains
lock together blocking the pores and effectively sealing
off the pores for solvent and ion conduction. Com-
putational analysis of the MscS structures suggests a
pore size of 4.9 ˚A in the closed state[2]. The presense
of hydrophobic side chains (lyseine rings) and the hy-
drophobic nature of TM3A itself in the pore area pre-
vent wetting of the pore in the closed state and block
conduction of ions and water molecules, and this is
called ’vapour lock’.
Further, particular residues have been identified that
are involved in the gating process, and decide when
the molecule will respond to osmotic pressure. Specif-
ically, A110 is found to move from one side of L115
to another during the movement of TM3a. It was hy-
pothesized and then proved by mutation studies that
the energetics of this movement are responsible for the
correct functioning of the gate - larger residues in place
of Alanine do not provide protection from downshock
since the energetics of opening are not favourable, and
smaller residues cause the gate to open prematurely.
Similarly, A102 was found to be importanat for gat-
ing, since it drags a methyl group across the TM3a
surface. Changing the energetics of this interaction by
mutation of A102 changes the gating behaviour. [2]
MscS is also capable of entering into a semiconduct-
ing state, which is observed at extreme osmotic pres-
sures which are capable of causing MscL to switch to
a conducting (open) state. This structure has not yet
been trapped and examined, however, and the struc-
tures that are known are believed to be the closed
and open forms of the the molecule. Another feature
of MscS is the ability called ’adaptation’, where the
molecule can switch to a deactivated state when os-
motic pressure returns to normal or may get desensi-
tized after prolonged or rapidly varying exposure. It
has been suggested that this can be explained based on
the fact that ’closing’ the gate involves the repacking of
the transmembrane helices and their side chains in the
intricate manner necessary for vapour lock. The rate of
adaptation of MscS has been found to be dependent on
protein-protein interactions between the various mov-
ing TM helices.[2]
The mechanism of gating itself is based on the un-
derstanding of the involvement of the transmembrane
helices in the gating process. It is believed that un-
der excessive outward turgor pressure, the decrease of
lipid density in the cytoplasm causes TM1 and TM2
to reorient themselves to increase their buried volume.
This movement of TM1 and TM2 acts as a lever on
TM3a, pulling it and its hydrophobic side chains away
from the pore, thereby causing it to open.
3 Structure based functional
understanding
The structure of MscS has been central to the under-
standing of its functioning in prokaryotic organisms,
and this is being used to model the channels known
to exist in eukayotes as well. While the existence of
these proteins and their biological function has been
known long before their structures were determined,
specific information about their mechanism of func-
tion has been largely unknown, and a number of ques-
tions still remain. The elucidation of their structures
has provided viatal information to answering questions
about how such channels function. Specifically, the
following are some of the insights that analysis of the
structure have led to :
• For a long time, it seems that the closed form
2

3. of the structure was believed to be the open
form. It seems that only after computational stud-
ies suggested that the hydrophobic residues on
TM3a would cause vapour lock in the pores, ef-
forts were redirected into isolating the open form
structure[2][3].
• The mechanism of gating, particularly the iris
like movement of transmembrane helices, was con-
firmed only after both the open and closed forms
of the structure were isolated and determined.[1]
• The availability of both open and closed form
structures has allowed computational analysis to
point to A110 and A102 as a potential ’important’
residues in the gating process, which was later con-
firmed by mutation studies.
• The process of ’adaptation’ has been given a pro-
posed mechanism only after the structures clearly
showed the hydrophobic finger network providing
the crucial ’vapor lock’ for gating.
4 Unanswered questions
Even after the elucidation of the structure of both the
closed and open form, a number of questions remain.
The understanding of mechanosensitive channels is still
in its early stages, where the mechanisms of action
are still largely hypothesis. The particular mechanisms
for gating and adaptation are still not clearly under-
stood. More details about the regulatory processes in-
volved in gating and adaptation, as well the semicon-
ducting state MscS is known to exhibit, are still to be
clearly understood. Questions regarding the assembly
of the channels also deserve study. In Eukaryotes, the
mechanosensitive channels have not yet been studied
in detail due to difficulties in isolating and crystalliz-
ing them, leaving a lot of open possibilities in their
mechanism and action.
5 Conclusions
Mechanosensitive channels such as MscS are important
for cell survival. Channels in general are a very im-
portant class of membrane proteins, and yet MscS is
one of the first channel proteins for which the struc-
ture is known for different configurations of the same
molecule, unlike in other cases where different config-
urations of the structure have been determined for ho-
mologs (such as for K+ ion channels). As such, de-
tails of the mechanisms involved in these proteins are
yet to be revealed. While it has taken two decades
to reach from the initial discovery of the existance
of mechanosensitive channels for the structure of the
open form to be revealed and reasonable hypothesis for
their mechanisms have been developed, it is expected
that the pace of development in this area is increasing
thanks to the improvement in tools and techniques for
isolation and analysis of proteins, their sequences and
structures both in in-vitro and in-vivo experiments as
well as using bioinfomatics approaches.
References
[1] Opening the Molecular Floodgates; Chris S. Gandhi
and Douglas C. Rees; Science 321, 1166 (2008); DOI:
10.1126/science.1162963
[2] The Structure of an Open Form of an E. coli Mechanosen-
sitive Channel at 3.45 ˚A Resolution; Wenjian Wang, et al.;
Science 321, 1179 (2008); DOI: 10.1126/science.1159262
[3] A Structural Mechanism for MscS Gating in Lipid Bilay-
ers; Valeria Vsquez, et al.; Science 321, 1210 (2008); DOI:
10.1126/science.1159674
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