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Mechanosensitive channels: an update
S18 Symposium 12: Moleculat and cellular biology of mechanosensitive ion channels. Chair: M. Sokabe (Japan)
s12-1 Mechanosensitive Channels: an update Sachs, F., Biophysics, SUNY, Buffalo, NY, USA 14214
Mechanosensitive (MS) ion channels are the least well studied of the major ion channel families. Much of the lag reflects and absence of pharmacological agents and controllable stimulators. While we have proposed that forces within the cytoskeleton transmit forces to the channels, the supporting data was always indirect. In contrast. MscL. a mechanosensitive ion channel~cloned from E. Co/i was reconstituted in active form in pure lipid bilayers showing that the cytoskeleton is not always required for mechanical sensitivity. How are the mechanical channels activated in eukaryotic cells? Recent experiments on the mechanics membrane patches from eukaryotic cells suggest that the lipids bear a significant fraction of the total tension, even in the absence of applied pressure. Activation of eukaryotic mechanosensitive ion channels may also arise from lipid tension. If tension reached channels through the lipids, it would explain why it is difficult to evoke whole cell MS currents with local stimulation. In whole cells, there is a large buffer of excess lipid that can flow into stressed regions thereby relaxing the tension. The cytoskeleton can modulate transduction by absorbing a fraction of the total stress. Consistent with the lipid pathway, the cloned MSCs TREK-l and TRAAK, as well as MscL are affected by amphiphiles with the effect depending, most likely, on the monolayer they populate. This work was supported by the NIH.
MscL and related mechanosensitive ion-channel proteins Ching Kong Laboratory of Molecular Biology, Universily of Wisconsin. Madison, S3706, U.S.A.
WI
The activity of mechanoSensitive &hannel of large conductance lMacL) wa\ first detected in a patch-clamp survey of the E. co/i surface 12 year? aeo. This activity remains when isolated membranes were reconstituted into artificial liposomes. By fractionating the active membrane extract. the corresponding protein was enriched and its gene cloned. Subsequent rtudies showed that it is a homomultimer of subunit, each wth two transmembrane a helices, and that it is opened by stretch forces from the lipid bilayer. A genetic dissection indicated the importance of the cytoplasmic end of the first helix in gatmg. Recent advances from five laboratories will be reviewed includtng (I) a crystal structure of MscL at atomic resolution; (2) a gene for E. m/i’s second MS channel and the hypotonic lysis phenotype of the douhlc mutant; (3) a calibration of the energetic and spatial parameters for MxL gating; and (4) a mutational analysis of a residue in the gate that trace< the path of channel opening.
S12-4
s12-2 The mechanosusceptibility
S12-3
of vdtagegated
ion channels.
Physiological role of cation selective signaling and their molecular entity.
MS channels
in cell
Maw& CL @ 6X. hanka Pf & Tubarm?N Neuroscience% Loeb Health Research institute, Ottawa Hospital, Ottawa Ontario, Canada KIY 4E9 Mechanosensitlve (MS) ion channels - those whose open probability changes with membrane tension - can be detected in almost all cells. Initially it was thought that MS channels were a specialized subgroup of ion channels, to be classed as “mechanogated” along with the ligand- and voltage-gated channels. We suggest that, apart from a few true mechanotrusducer channels, this view is incorrect and that specialized strucures are unnecessary for channels to show marked susceptibility to bilayer tension. This conclusion is based on our finding that the archetypical voltage-gated K channel, Shaker-IR (an inactivation-removed version), expressed in Xenopus oocytes and studied by patch clamp of macroscopic and of single channel currents, is as good a MS channel as any previously described. Shaker-IR exhibits reversible stretch-activation at voltages where its open probability is low and stretch-inactivation at voltages where its open probability is high. In addition, we have shown that the a-subunit (pore) of the human skeletal muscle voltage-gated Na channel, which, expressed in Xenopus oocytes, exhibits abnormal slowly-inactivating currents, converts irreversibly to the normal fast-inactivating form in response to membrane tension. The stretch-induced fast mode is kinetically indistinguishable from the fast mode obtained by co-expressing a and Sl subunits or by using high frequency stimulation of a alone. The widespread occurrence of mechanosusceptlble channels in patch recordings suggests that, in situ,but not in patches, channels are effectively mechanoprotected by the membrane cytoskeleton, possibly in conjunction with auxilliary channel subunits and/or regulatory proteins.
Sokabe M, Naruse K, Kojima I, and Iida H Department of Physiology, Nagoya University Medicine, Nagoya 466-8550, Japan
School
of
Cation selective mechanosensitive channels (MSCatCs) are most ubiquitously observed type in eucaryotic cells. However, their physiological role and molecular entity are not yet clear. We have found that stretch of human umbilical vein endothelial cells (HUVECs) increased intracellular Ca2’ concentration ([Ca”],), which was mostly caused by Ca2+ influx via activated MSCatCs expressed in HUVECs. We then analyzed the role of the stretchdependent [Ca”ll increase in the stretch-induced remodeling in HUVECs. When subjected to uniaxial cyclic stretch, cultured HUVECs started to change their morphology from a “cobble stone” appearance to a spidle-like shape aligning their longitude perpendicular to the stretch axis. This response was totally dependent on the stretch-induced [Ca”‘], increase, which drives the following signal cascade: [Ca’*]i increase -+ calcineurin -+ src+tyrosine phosphorylation of focal proteins -+ rearrangement of cytoskeletons and focal adhesions ---f cell remodeling. Such a critical role of MSCatCs was also observed in the cell volume regulation upon hypotonic stimulation to A6 cells. Thus we could say that the primary role of MSCatCs is to increase [Ca*‘]i. Very recently we have identified the gene, mid-l, that encodes a MSCatC in yeast cells. More rigorous test for the role of this channel and its structure-function study are in progress.
s12-1 Mechanosensitive Channels: an update Sachs, F., Biophysics, SUNY, Buffalo, NY, USA 14214
Mechanosensitive (MS) ion channels are the least well studied of the major ion channel families. Much of the lag reflects and absence of pharmacological agents and controllable stimulators. While we have proposed that forces within the cytoskeleton transmit forces to the channels, the supporting data was always indirect. In contrast. MscL. a mechanosensitive ion channel~cloned from E. Co/i was reconstituted in active form in pure lipid bilayers showing that the cytoskeleton is not always required for mechanical sensitivity. How are the mechanical channels activated in eukaryotic cells? Recent experiments on the mechanics membrane patches from eukaryotic cells suggest that the lipids bear a significant fraction of the total tension, even in the absence of applied pressure. Activation of eukaryotic mechanosensitive ion channels may also arise from lipid tension. If tension reached channels through the lipids, it would explain why it is difficult to evoke whole cell MS currents with local stimulation. In whole cells, there is a large buffer of excess lipid that can flow into stressed regions thereby relaxing the tension. The cytoskeleton can modulate transduction by absorbing a fraction of the total stress. Consistent with the lipid pathway, the cloned MSCs TREK-l and TRAAK, as well as MscL are affected by amphiphiles with the effect depending, most likely, on the monolayer they populate. This work was supported by the NIH.
MscL and related mechanosensitive ion-channel proteins Ching Kong Laboratory of Molecular Biology, Universily of Wisconsin. Madison, S3706, U.S.A.
WI
The activity of mechanoSensitive &hannel of large conductance lMacL) wa\ first detected in a patch-clamp survey of the E. co/i surface 12 year? aeo. This activity remains when isolated membranes were reconstituted into artificial liposomes. By fractionating the active membrane extract. the corresponding protein was enriched and its gene cloned. Subsequent rtudies showed that it is a homomultimer of subunit, each wth two transmembrane a helices, and that it is opened by stretch forces from the lipid bilayer. A genetic dissection indicated the importance of the cytoplasmic end of the first helix in gatmg. Recent advances from five laboratories will be reviewed includtng (I) a crystal structure of MscL at atomic resolution; (2) a gene for E. m/i’s second MS channel and the hypotonic lysis phenotype of the douhlc mutant; (3) a calibration of the energetic and spatial parameters for MxL gating; and (4) a mutational analysis of a residue in the gate that trace< the path of channel opening.
S12-4
s12-2 The mechanosusceptibility
S12-3
of vdtagegated
ion channels.
Physiological role of cation selective signaling and their molecular entity.
MS channels
in cell
Maw& CL @ 6X. hanka Pf & Tubarm?N Neuroscience% Loeb Health Research institute, Ottawa Hospital, Ottawa Ontario, Canada KIY 4E9 Mechanosensitlve (MS) ion channels - those whose open probability changes with membrane tension - can be detected in almost all cells. Initially it was thought that MS channels were a specialized subgroup of ion channels, to be classed as “mechanogated” along with the ligand- and voltage-gated channels. We suggest that, apart from a few true mechanotrusducer channels, this view is incorrect and that specialized strucures are unnecessary for channels to show marked susceptibility to bilayer tension. This conclusion is based on our finding that the archetypical voltage-gated K channel, Shaker-IR (an inactivation-removed version), expressed in Xenopus oocytes and studied by patch clamp of macroscopic and of single channel currents, is as good a MS channel as any previously described. Shaker-IR exhibits reversible stretch-activation at voltages where its open probability is low and stretch-inactivation at voltages where its open probability is high. In addition, we have shown that the a-subunit (pore) of the human skeletal muscle voltage-gated Na channel, which, expressed in Xenopus oocytes, exhibits abnormal slowly-inactivating currents, converts irreversibly to the normal fast-inactivating form in response to membrane tension. The stretch-induced fast mode is kinetically indistinguishable from the fast mode obtained by co-expressing a and Sl subunits or by using high frequency stimulation of a alone. The widespread occurrence of mechanosusceptlble channels in patch recordings suggests that, in situ,but not in patches, channels are effectively mechanoprotected by the membrane cytoskeleton, possibly in conjunction with auxilliary channel subunits and/or regulatory proteins.
Sokabe M, Naruse K, Kojima I, and Iida H Department of Physiology, Nagoya University Medicine, Nagoya 466-8550, Japan
School
of
Cation selective mechanosensitive channels (MSCatCs) are most ubiquitously observed type in eucaryotic cells. However, their physiological role and molecular entity are not yet clear. We have found that stretch of human umbilical vein endothelial cells (HUVECs) increased intracellular Ca2’ concentration ([Ca”],), which was mostly caused by Ca2+ influx via activated MSCatCs expressed in HUVECs. We then analyzed the role of the stretchdependent [Ca”ll increase in the stretch-induced remodeling in HUVECs. When subjected to uniaxial cyclic stretch, cultured HUVECs started to change their morphology from a “cobble stone” appearance to a spidle-like shape aligning their longitude perpendicular to the stretch axis. This response was totally dependent on the stretch-induced [Ca”‘], increase, which drives the following signal cascade: [Ca’*]i increase -+ calcineurin -+ src+tyrosine phosphorylation of focal proteins -+ rearrangement of cytoskeletons and focal adhesions ---f cell remodeling. Such a critical role of MSCatCs was also observed in the cell volume regulation upon hypotonic stimulation to A6 cells. Thus we could say that the primary role of MSCatCs is to increase [Ca*‘]i. Very recently we have identified the gene, mid-l, that encodes a MSCatC in yeast cells. More rigorous test for the role of this channel and its structure-function study are in progress.