Enzyme Domain Affects the Movement of the Voltage Sensor in Ascidian and Zebrafish Voltage-sensing Phosphatases

The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN). The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector. The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN). The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector. Voltage-gated ion channels play an important role in electrical activities and cell signaling of muscles and nerves. The first four transmembrane regions (S1-S4) are conserved among all the voltage-gated channels and operate as the voltage sensor, thus called the voltage sensor domain (VSD) 4The abbreviations used are: VSD, voltage sensor domain; VSP, voltage-sensing phosphatase; RT, reverse transcription; GST, glutathione S-transferase; NMDG, N-methyl-d-glucamine; MΩ, megohm; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-biphosphate; Ci-VSP, ascidian voltage-sensing phosphatase; Dr-VSP, D. rerio VSP; pC, picocoulomb; pF, picofarad. 4The abbreviations used are: VSD, voltage sensor domain; VSP, voltage-sensing phosphatase; RT, reverse transcription; GST, glutathione S-transferase; NMDG, N-methyl-d-glucamine; MΩ, megohm; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-biphosphate; Ci-VSP, ascidian voltage-sensing phosphatase; Dr-VSP, D. rerio VSP; pC, picocoulomb; pF, picofarad. (1Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (709) Google Scholar). The VSD regulates the operation of the downstream pore domain, consisting of the two transmembrane segments (S5-S6) and a loop that provides an ion permeation pathway. The VSD has several positively charged residues interspersed with two hydrophobic residues in the fourth transmembrane segment, called S4, that plays critical roles in voltage sensing (1Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (709) Google Scholar, 2Larsson H.P. Baker O.S. Dhillon D.S. Isacoff E.Y. Neuron. 1996; 16: 387-397Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 3Horn R. Ding S. Gruber H.J. J. Gen. Physiol. 2000; 116: 461-476Crossref PubMed Scopus (108) Google Scholar, 4Hille B. Ion Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland, MA2001Google Scholar). Recently resolution of the crystal structure of voltage-gated potassium channels (5Long S.B. Campbell E.B. MacKinnon R. Science. 2005; 309: 897-903Crossref PubMed Scopus (1809) Google Scholar, 6Long S.B. Tao X. Campbell E.B. MacKinnon R. Nature. 2007; 450: 376-382Crossref PubMed Scopus (1157) Google Scholar) and biophysical measurements of the movement of specific sites of the VSD (3Horn R. Ding S. Gruber H.J. J. Gen. Physiol. 2000; 116: 461-476Crossref PubMed Scopus (108) Google Scholar) have led to proposed models for the operation of the VSD (7Long S.B. Campbell E.B. MacKinnon R. Science. 2005; 309: 903-908Crossref PubMed Scopus (801) Google Scholar). However, the VSD has long been studied as a structure unique to voltage-gated ion channels. We have recently identified a protein, Ci-VSP, that contains the VSD but not the pore domain (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). Ci-VSP has the following two modules in its structure: the transmembrane spanning region that corresponds to the VSD of voltage-gated ion channels and the cytoplasmic region with homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN), a PtdIns(3,4,5)P3 phosphatase (9Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2550) Google Scholar). The VSD of Ci-VSP exhibits gating currents that indicate the conformational change in response to membrane voltage, and the phosphoinositide phosphatase activity is voltage-dependently regulated (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). This is the first example where an effector other than an ion pathway is regulated by the VSD. Furthermore, another protein that contains the VSD but lacks the pore domain was identified as the long sought molecular correlate of voltage-gated proton channels (10Sasaki M. Takagi M. Okamura Y. Science. 2006; 312: 589-592Crossref PubMed Scopus (411) Google Scholar, 11Ramsey I.S. Moran M.M. Chong J.A. Clapham D.E. Nature. 2006; 440: 1213-1216Crossref PubMed Scopus (445) Google Scholar). These findings indicate that the VSD is a protein module that operates as a self-contained functional unit. Recently, detailed relationships between voltage-sensor movement and phosphatase activity of Ci-VSP were examined by using several types of phosphoinositide sensors (12Murata Y. Okamura Y. J. Physiol. (Lond.). 2007; 583: 875-889Crossref Scopus (129) Google Scholar). This revealed that phosphatase activity changes over a range of membrane voltages as the voltage-sensor movement is increased. A shift of the voltage dependence of the VSD leads to a shift of the voltage dependence of the phosphatase activity to the same direction (12Murata Y. Okamura Y. J. Physiol. (Lond.). 2007; 583: 875-889Crossref Scopus (129) Google Scholar), verifying that VSD confers voltage sensitivity to phosphatase activities of the cytoplasmic region. Deletion of 8 amino acids in the linker region between the VSD and the phosphatase domain eliminates coupling between the two modules (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). The above findings indicate that VSD of VSP is tightly coupled with the downstream effector, as for voltage-gated ion channels. A recent study showed that after replacement of a part of the VSD of a voltage-gated potassium channel by that of Ci-VSP or a voltage-gated proton channel, the voltage-gating is still retained, indicating that the mechanisms of voltage sensing are conserved between conventional voltage-gated ion channels and voltage sensor domain proteins (13Alabi A.A. Bahamonde M.I. Jung H.J. Kim J.I. Swartz K.J. Nature. 2007; 450: 370-375Crossref PubMed Scopus (185) Google Scholar). Furthermore, a recent study with single molecule photobleaching has shown that Ci-VSP operates as a monomer (14Kohout S.C. Ulbrich M.H. Bell S.C. Isacoff E.Y. Nat. Struct. Mol. Biol. 2008; 15: 106-108Crossref PubMed Scopus (107) Google Scholar), indicating simpler stoichiometry than voltage-gated ion channels. Thus VSP serves as a simple model to understand how VSD is coupled with its effector. In this study, a VSP ortholog gene was identified from zebrafish, Danio rerio (named Dr-VSP). Dr-VSP shares most properties with Ci-VSP, including gating currents and voltage-sensitive phosphoinositide phosphatase activity. Dr-VSP showed much more robust gating currents in tsA201 cells than Ci-VSP, providing us with a unique opportunity to explore how the phosphatase module interacts with the VSD. We found that inhibition of phosphatase activity, either by the introduction of a point mutation or by pharmacological inhibition with vanadate, accelerates the movement of the VSD, providing evidence that the enzyme activity of the phosphatase domain influences the VSD movement. cDNAs and ortholog of Ci-VSP was cloned by KCNQ2/3 in the were by for The same were for cells. the VSP the VSP protein was used to by expressed were of the and region of VSP. on this two and and with from to as A was this the of VSP, and of were with the and These two were to the and the by of between and Dr-VSP was the between and of Ci-VSP, the regions of the and the (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar), were cloned and were using the in both were with in both of the modified The were by the with the used for the were and mutation of the and were The mutation was from using the following and mutation of the were and In of the phosphatase domain of Dr-VSP, was to the of the cytoplasmic region. The was by and and The protein was in and by The T. Dixon J.E. 2000; PubMed Scopus Google Scholar) was by using of and of and in the phosphatase following of protein was to and the was by The was by was to the and for The of were by measurements of was used for The of from and were by using a to the of and of Dr-VSP Dr-VSP was in the protein of the cytoplasmic region of A to the cytoplasmic region residues to was a that has a after the the protein was produced and from using a was from the cytoplasmic region of Dr-VSP by treatment with protein was and Dr-VSP were tsA201 cell with as a The were for to a membrane of was on The membrane was with the Dr-VSP and The was using and a of cells that were used for most of cells were used for of Dr-VSP with KCNQ2/3 because cells more robust currents than tsA201 cells. were in modified with in an with The cDNAs were using following the patch recording was using with tsA201 cells. The was The was NMDG, The and were with and and was were with and used as patch current were and currents were by to the gating was by of gating currents were examined by than with In gating currents were by single than a single was for the in many with the that more than single in the of the voltage sensor of Ci-VSP as examined by of of amino residues of in Ci-VSP (14Kohout S.C. Ulbrich M.H. Bell S.C. Isacoff E.Y. Nat. Struct. Mol. Biol. 2008; 15: 106-108Crossref PubMed Scopus (107) Google Scholar). The was by the where the is is is the and is the and in the have was not than 8 the was as as In this study, did not of in most of changes in gating current kinetics of than or gating currents than were not in with and robust gating currents of the were not in and were by from were by in for and on The was with with and was for an in a The were In was to by The the Dr-VSP was with and was used for of was a after The were in and PubMed Scopus Google Scholar). The was was after with current was by The for gating current was with The was and The The was to current was by a A was to for and for KCNQ2/3 currents were by a to The was were by with on a The were were with and as pervanadate was to the phosphatase activity of the phosphatase was in to was in a of and the was for was The was on used for was in to a of of orthovanadate, was to in the for patch and of showed that ortholog of Ci-VSP in of zebrafish, and a teleost Ci-VSP ortholog was using from to of zebrafish, D. The amino of VSP was used as a the of expressed a single of that the of the was The amino of the of Dr-VSP showed significant to Ci-VSP in the transmembrane regions and in the cytoplasmic and in the cytoplasmic Ci-VSP, Dr-VSP a of positively charged residues interspersed with hydrophobic residues in the transmembrane is a conserved among all voltage-gated ion channels. However, the of in Dr-VSP is distinct from Dr-VSP has a to of Ci-VSP The cytoplasmic phosphatase domain of VSP has a to that of in the phosphatase active This cysteine that is to the active of all of phosphoinositide phosphatase (9Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2550) Google Scholar) and to The downstream region of the cytoplasmic domain has homology to the domain of Ci-VSP, Dr-VSP lacks the domain is in the of whether the VSD of Dr-VSP membrane Ci-VSP (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar), gating currents as an of the movement of the voltage sensor (1Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (709) Google Scholar). A tsA201 cell was used as the of Dr-VSP cells produced robust ON- and OFF-gating currents and The and of the voltage showed that movement was between and over a range of membrane and with the that both and are from the same kinetics of and were examined distinct voltage by and of and are kinetics of a membrane were dependent on the of as more Ci-VSP was expressed in tsA201 cells to with gating currents of currents from Dr-VSP were more robust than from Ci-VSP was for Dr-VSP in The was more for Dr-VSP for than Ci-VSP The of Ci-VSP was than the of for Ci-VSP and Dr-VSP were and properties of voltage sensing of Ci-VSP are to in and gating in a In voltage-gated ion in play critical roles in voltage on are critical for the voltage-sensing of Dr-VSP, was by the residue, and gating currents were gating currents of were robust than for the Dr-VSP, the kinetics of ON- and were two of The was by was with the This is with the that a of Ci-VSP with replacement by a to of Dr-VSP, showed a shift of voltage dependence as shown by voltage (14Kohout S.C. Ulbrich M.H. Bell S.C. Isacoff E.Y. Nat. Struct. Mol. Biol. 2008; 15: 106-108Crossref PubMed Scopus (107) Google Scholar). In voltage dependence was than the for OFF-gating currents was for and with the that on to movement conformational change of the voltage the amino of Ci-VSP and Dr-VSP were was for Ci-VSP and for Dr-VSP the distinct voltage dependence of between Ci-VSP and Dr-VSP is from the amino in the VSD, was by and gating currents were In Dr-VSP was and the was as with the This has a to that of Ci-VSP We and this mutation kinetics and a shift of the not the shift was than that of These findings indicate that positively charged residues of play roles in voltage dependence of voltage sensor of VSP. is to phosphatidylinositol (9Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2550) Google Scholar), and Ci-VSP exhibits activity (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). A protein of cytoplasmic domain with glutathione of Dr-VSP was in and phosphatase activity was by the as (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). in a and from the point The of was as with of This was much than the PtdIns(3,4,5)P3 was with is a critical for phosphoinositide phosphatase activity (9Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2550) Google Scholar), and the to serine in Ci-VSP its phosphatase activity (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). was the cytoplasmic region of was with PtdIns(3,4,5)P3 the phosphatase activity of Dr-VSP is a to that for the of Ci-VSP (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). potassium channels were coexpressed with Dr-VSP in and voltage dependence was examined following the (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). The KCNQ2/3 potassium channel that current and is for its sensitivity to H. T. T. T. D.E. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar) was coexpressed with Dr-VSP, and current was membrane In cells that were with KCNQ2/3 the current a to following an for in and its with This is with (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar) and because of the of the voltage sensor of KCNQ2/3 channels. In that coexpressed Dr-VSP and the KCNQ2/3 potassium channel, the current of KCNQ2/3 channel with and with and did not change of KCNQ2/3 channel and as in that KCNQ2/3 channels Dr-VSP is a voltage-sensing phosphatase in the cytoplasmic phosphatase is regulated by the operation of the VSD as in This that a functional VSP is not to but is more in of by of the (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar), the of Ci-VSP the cytoplasmic region showed gating indicating that the VSD is a self-contained functional unit. In that it was that the kinetics of gating currents of the were than of the This on Ci-VSP us to gating currents from the of Dr-VSP the cytoplasmic region. of the currents were than the Dr-VSP between two modules is that kinetics of gating currents of the could to the of phosphatase activity. this gating currents were from the in in the active center of the phosphatase domain was to and showed kinetics in than of the of and were than of the and In with from the Dr-VSP, kinetics of of were of the movement of was is than that of was and for and was not and for the and that the protein is expressed as the protein, was A the cytoplasmic region of Dr-VSP a for the and of molecular changes in gating currents in the are a conserved among molecular of VSP, gating currents were from the of Ci-VSP with serine on cysteine that corresponds to cysteine in Dr-VSP and is to critical for phosphoinositide phosphatase activity (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). showed kinetics of than Ci-VSP indicating that of the voltage sensor movement in the is not to pharmacological inhibition of phosphatase activity of gating used pervanadate is to the activity of J. J. J. 1996; PubMed Scopus Google Scholar) by to a cysteine in the active center of phosphatases. was to the and than pervanadate treatment The and after of pervanadate The kinetics of as by of membrane is with pervanadate than its In the of of is than its over a range of membrane and not change with results with of pervanadate did not kinetics of gating currents of the Dr-VSP of gating currents by pervanadate is because of the of that was in pervanadate because of kinetics of gating currents was the and but not was used in the patch or to the not another of vanadate, orthovanadate, was in the patch of kinetics of gating currents was still and and of by the of to the of on of gating currents is to with mutation of in voltage-gated ion channels MacKinnon R. Neuron. 1996; 16: Full Text Full Text PDF PubMed Scopus Google Scholar, D. F. Neuron. 1996; 16: Full Text Full Text PDF PubMed Scopus Google Scholar). The above results with that on play a critical role in the movement of the voltage We the of of gating currents by the inhibition of phosphatase activity is with of gating currents by of were and that amino residues were interspersed with of hydrophobic This to as gating currents showed a shift of the to a direction currents were and after pervanadate the movement of gating of the is the was to In the of the is in a and after treatment with pervanadate were and In the kinetics of and was in the patch a shift of the was not currents were from with a mutation and with the The of the is to the direction as with that of the were and for the and the not These results indicate that of gating currents by the inhibition of phosphatase activity to the by the of in S4, indicating that mechanisms of of gating currents inhibition of phosphatase activity are distinct from with the of to the segments of the VSD. of by VSP of recent study (12Murata Y. Okamura Y. J. Physiol. (Lond.). 2007; 583: 875-889Crossref Scopus (129) Google Scholar) that depletion of is by the activity of is of the of with on of voltage sensor in voltage-gated ion channels has been to by on the of D. MacKinnon R. Nature. 2006; PubMed Scopus Google Scholar, Y. Y. Nature. 2008; PubMed Scopus Google Scholar), and the voltage sensor X. X. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of by phosphatase activity change the voltage-sensing movement of the VSD. the of kinetics of gating currents inhibition of enzyme activity is because of altered phosphoinositide examined kinetics of gating currents the of to depletion of to phosphatase activity of VSP We Dr-VSP by the activity of expressed KCNQ2/3 channels. cells with KCNQ2/3 channel and currents with of These properties are of KCNQ2/3 channels I.S. Dixon J.E. MacKinnon D. Science. 1998; PubMed Scopus Google Scholar). of KCNQ2/3 channel was by its sensitivity to a specific not to as the voltage over expressed with Dr-VSP, KCNQ2/3 current showed current the The of current were among cells by of the current the of a to the the of to by The were and for cells Dr-VSP and KCNQ2/3 and In most the a current as the voltage over or These are to reported for Ci-VSP in (12Murata Y. Okamura Y. J. Physiol. (Lond.). 2007; 583: 875-889Crossref Scopus (129) Google Scholar). In of current was much and to to These results indicate that current of KCNQ2/3 current as a of enzyme activity of Dr-VSP and that phosphatase activity of Dr-VSP by a to is to depletion a gating currents of Dr-VSP distinct kinetics between with state and gating currents were either with a of or a and of gating currents were of the Similar results were from two other cells. These findings that gating currents are not because of of distinct by altered phosphoinositide We that conformational change of the phosphatase module influences the movement of the VSD its In this study, the ortholog of VSP cloned from zebrafish, Dr-VSP, was expressed in cells. Dr-VSP robust gating us to study detailed kinetics distinct enzyme Dr-VSP showed kinetics of gating currents inhibition of phosphatase activity either by mutation or by We that conformational change of the phosphatase module influences the movement of the VSD the kinetics of voltage sensor movement on distinct enzyme of voltage-gated ion channels is to by on the of and the voltage sensor H. M. J. Physiol. (Lond.). Scopus Google Scholar, R. J. Biol. PubMed Scopus Google Scholar, J. Gen. Physiol. PubMed Scopus Google Scholar, J. J. Physiol. (Lond.). Scopus Google Scholar). have on of by the VSP phosphatase activity change the voltage sensor, movement of the VSD. However, this is for the following kinetics of gating currents was distinct of PtdIns(4,5)P2, were by of VSP phosphatase activities with or of In this change in kinetics and of gating currents was of pervanadate to cells Dr-VSP change of kinetics of gating currents were to where enzyme activity is not active (8Murata Y. Iwasaki H. Sasaki M. Inaba K. Okamura Y. Nature. 2005; 435: 1239-1243Crossref PubMed Scopus (534) Google Scholar). The cytoplasmic regions of VSP and a conserved structure called loop with The crystal structure of phosphatase showed that of loop is the loop in the of cysteine that a Rev. Mol. Biol. 1998; PubMed Scopus Google Scholar). In the of the phosphatase the of the the active enzyme center the loop changes the residue, is to structure is D. Science. PubMed Scopus Google Scholar, S. B. PubMed Scopus Google Scholar). In the ortholog a the of between the state and state of the loop the phosphatase active cysteine was to serine X. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Ci-VSP and Dr-VSP have a conserved in the region of the loop in Dr-VSP and in Ci-VSP, is that change of cysteine to the region to the loop of its or to a in the VSD more of other sites in the cytoplasmic domain that are to the VSD movement us a to understand how two protein modules are coupled in of voltage sensor movement by the cytoplasmic region coupling between VSD and its effector. The recently crystal of voltage-gated potassium channels (5Long S.B. Campbell E.B. MacKinnon R. Science. 2005; 309: 897-903Crossref PubMed Scopus (1809) Google Scholar) that VSD is a self-contained from the pore domain, the of the channel between voltage sensor movement and phosphatase activities that the voltage range of of enzyme activity with the range of movement (12Murata Y. Okamura Y. J. Physiol. (Lond.). 2007; 583: 875-889Crossref Scopus (129) Google Scholar). This that the VSD of VSP is not a simple that confers to constitutively active but operates as a to of voltage sensor movement by the enzyme domain that the VSD is tightly to the the above In the voltage-gated potassium channel MacKinnon R. Neuron. 1996; 16: Full Text Full Text PDF PubMed Scopus Google Scholar), a model has been proposed in the linker that the voltage sensor and pore domain channel (7Long S.B. Campbell E.B. MacKinnon R. Science. 2005; 309: 903-908Crossref PubMed Scopus (801) Google Scholar). of the channel more in the of in the pore than in the of D. D. J. Gen. Physiol. PubMed Scopus Google Scholar), that a conformational change in the pore domain could to the state of voltage sensor movement in voltage-gated ion channels. of how mechanisms of between the VSD and its effector are distinct or between and voltage-gated ion channels on the of voltage sensor We for on with KCNQ2/3 and and R. for critical with

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