Kazuaki Homma

Optical stimulation has enabled important advances in the study of brain function and other biological processes, and holds promise for medical applications ranging from hearing restoration to cardiac pace making. In particular, pulsed laser stimulation using infrared wavelengths >1.5 μm has therapeutic potential based on its ability to directly stimulate nerves and muscles without any genetic or chemical pre-treatment. However, the mechanism of infrared stimulation has been a mystery, hindering its path to the clinic. Here we show that infrared light excites cells through a novel, highly general electrostatic mechanism. Infrared pulses are absorbed by water, producing a rapid local increase in temperature. This heating reversibly alters the electrical capacitance of the plasma membrane, depolarizing the target cell. This mechanism is fully reversible and requires only the most basic properties of cell membranes. Our findings underscore the generality of pulsed infrared stimulation and its medical potential. Pulsed infrared laser light can directly stimulate nerves and muscles, but the underlying biophysical mechanism has remained enigmatic. This study reveals that infrared pulses depolarize target cells by reversibly altering the electrical capacitance of the plasma membrane.

Nitric oxide/cGMP pathway induces vasodilatation, yet the underlying mechanism is obscure. In the present study, we studied the mechanism of cGMP-induced relaxation of the smooth muscle contractile apparatus using permeabilized rabbit femoral arterial smooth muscle. 8-Br-cGMP–induced relaxation was accompanied with a decrease in myosin light chain (MLC) phosphorylation. MLC phosphatase (MLCP) activity, once decreased by agonist-stimulation, recovered to the resting level on addition of 8-Br-cGMP. Because MLCP activity is regulated by the phosphorylation of a MLCP-specific inhibitor, CPI17 at Thr38 and MBS (myosin binding subunit of MLCP) at Thr696, we examined the effect of 8-Br-cGMP on the phosphorylation of these MLCP modulators. Whereas CPI17 phosphorylation was unchanged after addition of 8-Br-cGMP, MBS phosphorylation at Thr696 was significantly decreased by 8-Br-cGMP. We found that 8-Br-cGMP markedly increased MBS phosphorylation at Ser695 in the fiber pretreated with phenylephrine. MBS phosphorylation of Thr696 phosphorylated MBS at Ser695 partially resumed MLCP activity inhibited by Thr696 phosphorylation. Whereas Ser695 phosphorylation was markedly increased, the extent of diphosphorylated MBS at Ser695 and Thr696 in fibers was unchanged after cGMP-stimulation. We found that MBS phosphatase activity in arteries for both diphosphorylated MBS and monophosphorylated MBS at Thr696 significantly increased by 8-Br-cGMP, whereas MBS kinase activity was unchanged. These results suggest that the phosphorylation at Ser640 induced by cGMP shifted the equilibrium of the Thr641 phosphorylation toward dephosphorylation, thus increasing MLCP activity. This results in the decrease in MLC phosphorylation and smooth muscle relaxation.

Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca2+ concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca2+ binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain. Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca2+ concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca2+ binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain. 4-morpholinepropanesulfonic acid Myosins are motor proteins that translocate actin filaments upon hydrolysis of ATP, and thus they play a critical role in diverse forms of cell contractility and motility. During the last decade a number of myosin-like proteins have been found, and the myosins are currently organized into 15 classes based upon phylogenetic sequence comparisons of the motor domains (1Cheney R.E. Mooseker M.S. Curr. Opin. Cell Biol. 1992; 4: 27-35Crossref PubMed Scopus (334) Google Scholar, 2Cheney R.E. Riley M.A. Mooseker M.S. Cell Motil. Cytoskeleton. 1993; 24: 215-223Crossref PubMed Scopus (236) Google Scholar, 3Goodson H.V. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 659-663Crossref PubMed Scopus (140) Google Scholar, 4Mooseker M.S. Cheney R.E. Annu. Rev. Cell Dev. Biol. 1995; 11: 633-675Crossref PubMed Scopus (386) Google Scholar, 5Titus M.A. Trends Cell Biol. 1997; 7: 119-123Abstract Full Text PDF PubMed Scopus (53) Google Scholar). Class V myosin was originally identified in brain as a calmodulin-binding protein that had actin-dependent ATPase activity (6Espindola E.M. Espreafico E.M. Coelho M.V. Martins A.R. Costa F.R.C. Mooseker M.S. Larson R.E. J. Cell Biol. 1992; 118: 359-368Crossref PubMed Scopus (99) Google Scholar). Myosin V is a member of the myosin superfamily that is expressed in variety of cell types and is involved in a variety of membrane trafficking and organelle transport functions (1Cheney R.E. Mooseker M.S. Curr. Opin. Cell Biol. 1992; 4: 27-35Crossref PubMed Scopus (334) Google Scholar, 2Cheney R.E. Riley M.A. Mooseker M.S. Cell Motil. Cytoskeleton. 1993; 24: 215-223Crossref PubMed Scopus (236) Google Scholar, 3Goodson H.V. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 659-663Crossref PubMed Scopus (140) Google Scholar, 4Mooseker M.S. Cheney R.E. Annu. Rev. Cell Dev. Biol. 1995; 11: 633-675Crossref PubMed Scopus (386) Google Scholar, 5Titus M.A. Trends Cell Biol. 1997; 7: 119-123Abstract Full Text PDF PubMed Scopus (53) Google Scholar). Myosin V has two heads that are connected with a long coiled-coil domain; however, in contrast to conventional myosin, it contains a globular C-terminal domain and does not form thick filaments (7Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar). The head domain is composed of a globular motor domain and an elongated neck domain that is associated with a number of light chains. The sequence at the neck region contains six IQ motifs that have been implicated as calmodulin or myosin light chain binding consensus motifs as found in a variety of calmodulin-binding proteins and myosins (7Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar). Since light chains play a critical role in the regulation of various conventional myosins, it has been proposed that the IQ domain serves as a regulatory component of myosin V. The role of the IQ motif and bound calmodulin serving as a regulatory component of unconventional myosins is best studied for mammalian myosin Is. For both brush border myosin I (8Collins K. Sellers J.R. Matsudaira P.T. J. Cell Biol. 1990; 110: 1137-1147Crossref PubMed Scopus (145) Google Scholar, 9Swanljung-Colling H. Colling J.H. J. Biol. Chem. 1991; 266: 1312-1319Abstract Full Text PDF PubMed Google Scholar, 10Wolenski J.S. Hayden S.M. Forscher P. Mooseker M.S. J. Cell Biol. 1993; 122: 613-621Crossref PubMed Scopus (66) Google Scholar) and myosin Iβ (11Barylko B. Wagner M.C. Reizes O. Albanesi J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 90: 490-494Crossref Scopus (78) Google Scholar, 12Zhu T. Ikebe M. Biochemistry. 1996; 35: 513-522Crossref PubMed Scopus (67) Google Scholar, 13Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), high Ca2+ inhibits motor activity due to Ca2+binding to the calmodulin light chain. Since 1 mol of bound calmodulin dissociates from myosin I at high Ca2+, it was originally thought that this dissociation of calmodulin was responsible for the inhibition of myosin I motor activity. However, since virtually no calmodulin dissociation is observed at pCa 6 where the motility activity is completely abolished, this view has been questioned (13Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). For naturally isolated myosin V, motility activity is inhibited at high Ca2+, whereas actin-activated ATPase activity markedly increases in the presence of Ca2+ (7Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (379) Google Scholar). Recently it was shown (14Trybus K.M. Kremensova E. Freyzon Y. J. Biol. Chem. 1999; 274: 27448-27456Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) that a truncated recombinant monomeric myosin V with two IQ motifs had motility activity that was inhibited at high Ca2+, but only in the absence of exogenous calmodulin, suggesting that the inhibition is via the physical dissociation of calmodulin. Interestingly, the truncated myosin V showed inhibition of actin-activated ATPase activity by Ca2+ rather than activation, as is found for naturally isolated myosin V (14Trybus K.M. Kremensova E. Freyzon Y. J. Biol. Chem. 1999; 274: 27448-27456Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). This apparent discrepancy is not understood, but there are several possible explanations. Since the two-headed structure is critical for the regulation of both conventional smooth muscle and non-muscle myosin motor function, it is plausible that the two-headed structure may play some role in the regulatory mechanisms of myosin V. Alternatively, a complete neck domain may be required for the proper regulation of myosin V. The aim of the present study is to clarify the regulation of myosin V motor function by Ca2+. To address these questions, we have produced a two-headed myosin V construct, a single-headed construct having the entire neck domain (six IQ motifs), and a truncated single-headed construct containing only two IQ motifs. These myosin V constructs were expressed, purified, and examined for motor function. It was found that although calmodulin dissociation is responsible for inhibition of actin-activated ATPase activity at high Ca2+, inhibition of motility occurs at lower Ca2+ where calmodulin is not dissociated from myosin V heavy chain. Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, MA). Actin was prepared from rabbit skeletal muscle acetone powder according to Spudich and Watt (15Spudich J.A. Watt J. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). Recombinant calmodulin from Xenopus oocyte (16Chien Y. Dawid I. Mol. Cell. Biol. 1984; 4: 507-513Crossref PubMed Scopus (118) Google Scholar) was expressed in Escherichia coli as described (17Ikebe M. Kambara T. Stafford W.F. Sata M. Katayama E. Ikebe R. J. Biol. Chem. 1998; 273: 17702-17707Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). A baculovirus transfer vector for mouse myosin V variants in pBluebac4 (Invitrogen, CA) was produced as follows. Mouse myosin V cDNA clones containing −35–1549 and 1549–3928 in pBluescript were kindly supplied by Dr. N. Jenkins (NCI, National Institutes of A cDNA with was to the vector containing a cDNA at the site at A site was at the of the a site at was the acid and a site was at A cDNA was with and to a baculovirus transfer vector containing a sequence with a at the of the This construct containing the entire coiled-coil was to double-headed myosin V. To single-headed myosin V with complete IQ site was at of The vector was with and the was The vector was and as a construct a single-headed myosin V with complete IQ motifs A site was at or of containing and of myosin V were to single-headed myosin V that two IQ motifs and one IQ motif (M5IQ1), For a sequence were at of the To recombinant myosin V, of Sf9 1 were with two the myosin V heavy chain and calmodulin, The were at in and were with in of 2 2 and 2 at for 15 the was with and at for to completely was to the and to the expressed myosin V. The were with A and with A with and to myosin V from The was with of in a a for at The was a and was with of and Myosin V was with and containing myosin V were and and 1 The myosin V was and 2 1 of isolated myosin V was was a the of PubMed Scopus Google Scholar). were smooth muscle myosin heavy chain myosin regulatory light chain and The of the myosin V heavy chain and calmodulin was by as described (13Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The ATPase activity was by at as described M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The in motility was as described M. M. Ikebe M. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). Myosin V was to the For a was with and the of the was with Myosin V was to the Actin was from the and the in was for of A of was to be to study the regulatory of myosin V motor function, various myosin V constructs were produced and expressed in Sf9 cells. The DHM5 construct contains the entire coiled-coil domain in addition to the complete head it is that it has a double-headed structure SHM5 has an entire head domain with complete IQ motifs but no coiled-coil thus it is to be a single-headed the M5IQ2 contains a motor domain but only 2 IQ motifs of the six IQ motifs constructs to in The were at the C-terminal of the rather than to possible of the protein and motor function. of a at the C-terminal of myosins has been with conventional J. Ikebe R. K. Ikebe M. Biochemistry. PubMed Scopus Google Scholar) as as unconventional myosin, and no motor function was The were with myosin and The of the two to the best myosin V was It be that myosin V with bound calmodulin be the of but that the of myosin V increases with calmodulin This was for constructs having an entire IQ of the myosin V construct was composed of a high and a The of was with the of myosin construct, and for and These were by CA) that these high are the expressed myosin V heavy chains The showed a with a in that is of calmodulin, suggesting that the are calmodulin. The of the was The of calmodulin myosin V heavy chain for and M5IQ2 were and This is with the number of IQ motifs in of myosin V The motor domain of myosin V is by light chains are The coiled-coil domain is by of myosin V are to the of myosin V construct was with the by the acid of by Ca2+. and are 5, and are in 1 Ca2+, and 6, are in 1 2 and 6, and The actin-activated ATPase activity was as a function of Ca2+ For and the ATPase activity markedly M5IQ1 showed no pCa Interestingly, inhibition of ATPase activity was reversed by addition of calmodulin in The of exogenous calmodulin the ATPase activity was for all the calmodulin of the actin-activated ATPase activity of the myosin V The calmodulin the of the ATPase activity for the constructs was and a was observed at was no in calmodulin of ATPase activity pCa and suggesting that the Ca2+ is the of the actin-activated ATPase of the The actin was to for the from the actin of the ATPase activity was and for SHM5 and in the presence of the the actin at high showed two At concentration, activity with actin concentration, and it was not from that in the presence of However, the activity at actin This that the inhibition of ATPase activity is not due to a in the affinity for The binding of myosin V constructs to was examined by in the presence of to the of Ca2+ the affinity myosin V and For all constructs a of myosin V with at than but no of Ca2+ the binding was observed The in of the ATPase of myosin V at high and is of the actin-activated ATPase activity of myosin V The actin-activated ATPase activity of myosin V constructs was as described in exogenous calmodulin was pCa The of the ATPase activity inhibition by high of exogenous calmodulin that it is due to a in the affinity of calmodulin for myosin V calmodulin Ca2+. To address this the dissociation of calmodulin from myosin V was Myosin V constructs were with in various Ca2+ and to bound calmodulin. myosin V with bound calmodulin was by and the calmodulin was by with myosin V heavy chain. a calmodulin was with but no calmodulin was detected. Myosin V constructs were for but no myosin V was in the absence of shown in the calmodulin bound to myosin V pCa 6 and for all of bound calmodulin was dissociated from M5IQ2 heavy that 1 mol of calmodulin is dissociated from the of the bound calmodulin was dissociated from the DHM5 and SHM5 that 2 mol of bound calmodulin of 6 are the motility activity of DHM5 and M5IQ2 at various with the actin-activated ATPase the motility activity was completely inhibited at high is that the inhibition of motility was at lower Ca2+ than the inhibition of the ATPase activity. in contrast to the inhibition of ATPase addition of exogenous calmodulin not the motility inhibition and exogenous calmodulin to at pCa but the motility activity was not in contrast to the ATPase activity. A was at pCa The of motility inhibited at high Ca2+ was only by A and It was shown that the is the for the actin-activated ATPase of truncated myosin V E.M. E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). inhibition of the ATPase activity of myosin V and the of Ca2+ this inhibition were shown in inhibited the actin-activated ATPase activity both in the presence and absence of Ca2+. a of E.M. E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), a of from the of the ATPase activity whereas a of found pCa This was lower than for conventional myosins B. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, B. Biochemistry. 1997; PubMed Scopus Google Scholar). Interestingly, Ca2+ not the inhibition that the affinity of myosin V for is not by Ca2+. for muscle myosin the myosins have motor function or regulatory the myosin The motor function of conventional myosin from smooth muscle and non-muscle is by the of regulatory light chain of the New Scholar, J.R. Scopus Google Scholar, S. Spudich J.A. Annu. Rev. 1992; PubMed Scopus Google Scholar). conventional myosin is by Ca2+ binding to the light chain as by the regulatory light chain I. 1995; Scopus Google Scholar). Interestingly, the regulation a two-headed structure and the of in the regulatory M. M. Ikebe M. Biochemistry. 1996; 35: PubMed Scopus Google Scholar, Sellers J.R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Ikebe M. 1995; PubMed Scopus Google Scholar). Myosin I regulation is the studied of the unconventional myosins from These myosin calmodulin as light and Ca2+ binding to this calmodulin light chain motor activity (8Collins K. Sellers J.R. Matsudaira P.T. J. Cell Biol. 1990; 110: 1137-1147Crossref PubMed Scopus (145) Google Scholar, 9Swanljung-Colling H. Colling J.H. J. Biol. Chem. 1991; 266: 1312-1319Abstract Full Text PDF PubMed Google Scholar, 10Wolenski J.S. Hayden S.M. Forscher P. Mooseker M.S. J. Cell Biol. 1993; 122: 613-621Crossref PubMed Scopus (66) Google Scholar, B. Wagner M.C. Reizes O. Albanesi J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 90: 490-494Crossref Scopus (78) Google Scholar, 12Zhu T. Ikebe M. Biochemistry. 1996; 35: 513-522Crossref PubMed Scopus (67) Google Scholar, 13Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). These results several the regulation of myosin V. Ca2+ myosin V motor does calmodulin play a critical role in the the number of in the myosin V for the is the two-headed structure critical for this we produced constructs of myosin V to address these double-headed myosin V, single-headed myosin V with complete IQ and myosin V with two IQ motifs. It was that the light chain of conventional myosin with myosin V from Cheney R.E. S.M. M.S. Mol. Biol. Cell. 1996; 7: Scholar) suggesting that it as a of myosin V. However, the light chain was not found in a myosin V from it was that myosin V expressed in Sf9 that were with calmodulin and light chain contains calmodulin and not the light chain O. B. J.A. Sellers J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the myosin V variants produced in the present study that have only calmodulin as light chains the of myosin V. double-headed and single-headed myosin V constructs to produced in the present study were O. B. J.A. Sellers J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It was that the double-headed myosin V was and cell by at the IQ domain O. B. J.A. Sellers J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we have not of double-headed myosin V the is since the was found at the IQ domain it is that the due to a of calmodulin binding with this the SHM5 isolated by Cheney R.E. S.M. M.S. Mol. Biol. Cell. 1996; 7: Scholar) in ATPase activity with exogenous calmodulin in suggesting that SHM5 was not with calmodulin. The myosin V constructs in this study were not by exogenous calmodulin suggesting that the are with bound calmodulin with the of the bound calmodulin. of these myosin V and showed the same pCa in actin-activated ATPase activity. The activity was above pCa It be that this in the ATPase activity is due to the dissociation of calmodulin from the heavy chain myosin calmodulin and and 1 mol of calmodulin from M5IQ2 or mol of calmodulin from DHM5 and SHM5 were dissociated from the heavy and the in ATPase activity observed at pCa and was completely reversed by addition of exogenous calmodulin. Since Ca2+ to calmodulin this of Ca2+, the be that Ca2+ binding to the calmodulin bound to myosin V the affinity of calmodulin for myosin V. However, since only 1 or 2 mol of the bound calmodulin dissociates from myosin V, the apparent in affinity be the The of the binding of the IQ with calmodulin has been A. M. 1996; 4: Full Text Full Text PDF PubMed Scopus Google Scholar). the absence of Ca2+, the of calmodulin a whereas the C-terminal a that with the of the IQ via a number of Ca2+ calmodulin the thus the C-terminal to the IQ the and the IQ may may in a to the IQ since in it the found in various calmodulin M. A. 1992; PubMed Scopus Google Scholar). It is that the dissociation of calmodulin at high Ca2+ occurs at IQ that not calmodulin to at high Ca2+ due to a of The present that mol of calmodulin are dissociated from myosin V at high Ca2+. site be the IQ since M5IQ2 1 mol of calmodulin at high M5IQ1 does This is with the (14Trybus K.M. Kremensova E. Freyzon Y. J. Biol. Chem. 1999; 274: 27448-27456Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). possible site is the last IQ is and where the consensus that via with the of calmodulin to the A. M. 1996; 4: Full Text Full Text PDF PubMed Scopus Google is by It is of to Ca2+ the actin-activated ATPase activity. Actin binding of myosin V in the presence of was with Ca2+ does not affinity for It was J. Ikebe R. K. Ikebe M. Biochemistry. PubMed Scopus Google Scholar) that the of the truncated myosin V construct, (M5IQ1), is the examined the of the inhibition of the ATPase but the apparent was not by Ca2+. Interestingly, an inhibition of ATPase activity was observed at but not in the presence of Ca2+. For conventional myosin it is that the inhibition of ATPase activity by high is due to the inhibition of the hydrolysis by A.R. 1992; PubMed Scopus Google Scholar). it is plausible that the inhibition of the ATPase activity by Ca2+ for myosin V is due to an inhibition of hydrolysis The inhibition of the actin-activated ATPase activity was completely reversed by the presence of of calmodulin. Since a of calmodulin of several is present in the inhibition of ATPase activity may not be in is that the motility of myosin V is completely pCa 6 where no apparent dissociation of calmodulin addition of a high of exogenous calmodulin to this inhibition of motility. These results indicate that the inhibition of the motility of myosin V is not due to dissociation of calmodulin from the heavy chain. The inhibition occurs pCa and 6 where Ca2+ is in cell the observed inhibition is results were for mammalian myosin Iβ T. Ikebe M. Biochemistry. 1996; 35: 513-522Crossref PubMed Scopus (67) Google Scholar, 13Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Since this of Ca2+ to the high affinity C-terminal of calmodulin, it is that Ca2+ binding to the C-terminal of calmodulin and conformational changes are responsible for the inhibition of motility. this it was shown that of the C-terminal inhibition of the motility of myosin Iβ (13Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). it was that truncated myosin V containing 2 IQ motifs showed motility activity at high Ca2+ high exogenous calmodulin was present although the of the motility is (14Trybus K.M. Kremensova E. Freyzon Y. J. Biol. Chem. 1999; 274: 27448-27456Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). At present the of this apparent discrepancy the present and that by (14Trybus K.M. Kremensova E. Freyzon Y. J. Biol. Chem. 1999; 274: 27448-27456Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) is The of the motility at high Ca2+ was not due to the of myosin V since the of the cell with Ca2+ or completely the motility examined various exogenous calmodulin to that is than that by (14Trybus K.M. Kremensova E. Freyzon Y. J. Biol. Chem. 1999; 274: 27448-27456Abstract Full Text Full Text PDF PubMed Scopus (101) Google it is that the of the motility is due to the of the bound calmodulin. the inhibition of the motility is observed at pCa 6, where virtually no calmodulin is dissociated from myosin V heavy chain. The present results that regulation of motility two IQ in the conformational of calmodulin, but not the physical dissociation of calmodulin, the inhibition of motility. is an apparent the hydrolysis and at Ca2+. This is from the regulation of conventional myosins in the regulatory domain both ATPase and of the New Scholar, J.R. Scopus Google Scholar, S. Spudich J.A. Annu. Rev. 1992; PubMed Scopus Google Scholar, I. 1995; Scopus Google Scholar). It is plausible that the in calmodulin the of the and thus the and Alternatively, the conformational of calmodulin the calmodulin and the domain of myosin V, thus motility. The present results indicate that the two-headed structure is not critical for regulation of activity of myosin V. This is from the regulation of conventional myosin, in an the two heads is involved in the regulation M. M. Ikebe M. Biochemistry. 1996; 35: PubMed Scopus Google Scholar, J. Ikebe R. K. Ikebe M. Biochemistry. PubMed Scopus Google Scholar, I. 1995; Scopus Google Scholar, Sellers J.R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Ikebe M. 1995; PubMed Scopus Google Scholar). myosin a single-headed unconventional myosin having calmodulin as light chain has been shown to have Ca2+ to that shown for myosin V in the present study (8Collins K. Sellers J.R. Matsudaira P.T. J. Cell Biol. 1990; 110: 1137-1147Crossref PubMed Scopus (145) Google Scholar, 9Swanljung-Colling H. Colling J.H. J. Biol. Chem. 1991; 266: 1312-1319Abstract Full Text PDF PubMed Google Scholar, 10Wolenski J.S. Hayden S.M. Forscher P. Mooseker M.S. J. Cell Biol. 1993; 122: 613-621Crossref PubMed Scopus (66) Google Scholar, B. Wagner M.C. Reizes O. Albanesi J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 90: 490-494Crossref Scopus (78) Google Scholar, 12Zhu T. Ikebe M. Biochemistry. 1996; 35: 513-522Crossref PubMed Scopus (67) Google Scholar, 13Zhu T. Beckingham K. Ikebe M. J. Biol. Chem. 1998; 273: 20481-20486Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). it is plausible that there is a motor activity regulatory in unconventional myosins calmodulin light chains. However, and is required for of the Dr. J. of for the Dr. H. for Dr. Jenkins for mouse myosin V