Ca2+ Activation of Smooth Muscle Contraction

Smooth muscle contraction is activated by phosphorylation of the 20-kDa light chains of myosin catalyzed by Ca2+/calmodulin (CaM)-dependent myosin light chain kinase (MLCK). According to popular current theory, the CaM involved in MLCK regulation is Ca2+-free and dissociated from the kinase at resting cytosolic free Ca2+concentration ([Ca2+]i). An increase in [Ca2+]i saturates the four Ca2+-binding sites of CaM, which then binds to and activates actin-bound MLCK. The results of this study indicate that this theory requires revision. Sufficient CaM was retained after skinning (demembranation) of rat tail arterial smooth muscle in the presence of EGTA to support Ca2+-evoked contraction, as observed previously with other smooth muscle tissues. This tightly bound CaM was released by the CaM antagonist trifluoperazine (TFP) in the presence of Ca2+. Following removal of the (Ca2+)4-CaM-TFP2 complex, Ca2+ no longer induced contraction. The addition of exogenous CaM to TFP-treated tissue at a [Ca2+] subthreshold for contraction or even in the absence of Ca2+(presence of 5 mm EGTA), followed by washout of unbound CaM, restored Ca2+-induced contraction; this required MLCK activation, since it was blocked by the MLCK inhibitor ML-9. The data suggest, therefore, that a specific pool of cellular CaM, tightly bound to myofilaments at resting [Ca2+]i, or even in the absence of Ca2+, is responsible for activation of contraction following a local increase in [Ca2+]. This mechanism would allow for localized changes in [Ca2+] in regions of the cell distant from the myofilaments to regulate distinct Ca2+-dependent processes without triggering a contractile response. Immobilized CaM, therefore, resembles troponin C, the Ca2+-binding regulatory protein of striated muscle, which is also bound to the thin filament in a Ca2+-independent manner. Smooth muscle contraction is activated by phosphorylation of the 20-kDa light chains of myosin catalyzed by Ca2+/calmodulin (CaM)-dependent myosin light chain kinase (MLCK). According to popular current theory, the CaM involved in MLCK regulation is Ca2+-free and dissociated from the kinase at resting cytosolic free Ca2+concentration ([Ca2+]i). An increase in [Ca2+]i saturates the four Ca2+-binding sites of CaM, which then binds to and activates actin-bound MLCK. The results of this study indicate that this theory requires revision. Sufficient CaM was retained after skinning (demembranation) of rat tail arterial smooth muscle in the presence of EGTA to support Ca2+-evoked contraction, as observed previously with other smooth muscle tissues. This tightly bound CaM was released by the CaM antagonist trifluoperazine (TFP) in the presence of Ca2+. Following removal of the (Ca2+)4-CaM-TFP2 complex, Ca2+ no longer induced contraction. The addition of exogenous CaM to TFP-treated tissue at a [Ca2+] subthreshold for contraction or even in the absence of Ca2+(presence of 5 mm EGTA), followed by washout of unbound CaM, restored Ca2+-induced contraction; this required MLCK activation, since it was blocked by the MLCK inhibitor ML-9. The data suggest, therefore, that a specific pool of cellular CaM, tightly bound to myofilaments at resting [Ca2+]i, or even in the absence of Ca2+, is responsible for activation of contraction following a local increase in [Ca2+]. This mechanism would allow for localized changes in [Ca2+] in regions of the cell distant from the myofilaments to regulate distinct Ca2+-dependent processes without triggering a contractile response. Immobilized CaM, therefore, resembles troponin C, the Ca2+-binding regulatory protein of striated muscle, which is also bound to the thin filament in a Ca2+-independent manner. The mechanism of activation of smooth muscle contraction is fundamentally different from that of skeletal and cardiac (striated) muscles, although both are triggered by an increase in cytosolic free Ca2+ concentration ([Ca2+]i) (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1733) Google Scholar, 2Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1342) Google Scholar). In striated muscles, Ca2+ binds to troponin C, inducing a conformational change in proteins of the contractile machinery, ultimately enabling actin-myosin interaction and cross-bridge cycling. Smooth muscle cells do not express troponin. Instead, Ca2+binds to CaM, 1The abbreviations and trivial name used are: CaM, calmodulin; H-T, Hepes-Tyrode; MLCK, myosin light chain kinase; RTA, rat tail artery; SR, sarcoplasmic reticulum; TFP, trifluoperazine; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; ML-9, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine-HCl. a homolog of troponin C (3Nakayama S. Kretsinger R.H. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 473-507Crossref PubMed Scopus (182) Google Scholar). According to current theory (Fig.1A), at resting [Ca2+]i, the CaM involved in contraction is Ca2+-free and cytosolic (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1733) Google Scholar, 4Allen B.G. Walsh M.P. Trends Biochem. Sci. 1994; 19: 362-368Abstract Full Text PDF PubMed Scopus (163) Google Scholar, 5Kamm K.E. Stull J.T. J. Biol. Chem. 2001; 276: 4527-4530Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 6Zimmermann B. Somlyo A.V. Ellis-Davies G.C.R. Kaplan J.H. Somlyo A.P. J. Biol. Chem. 1995; 270: 23966-23974Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Following an increase in [Ca2+]i, Ca2+ binds to the four Ca2+-binding sites of CaM, which then interacts with and activates MLCK that is tethered via its N terminus to actin (7Dabrowska R. Hinkins S. Walsh M.P. Hartshorne D.J. Biochem. Biophys. Res. Commun. 1982; 107: 1524-1531Crossref PubMed Scopus (48) Google Scholar, 8Kanoh S. Ito M. Niwa E. Kawano Y. Hartshorne D.J. Biochemistry. 1993; 32: 8902-8907Crossref PubMed Scopus (42) Google Scholar, 9Lin P. Luby-Phelps K. Stull J.T. J. Biol. Chem. 1999; 274: 5987-5994Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 10Smith L. Su X. Lin P. Zhi G. Stull J.T. J. Biol. Chem. 1999; 274: 29433-29438Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 11Smith L. Stull J.T. FEBS Lett. 2000; 480: 298-300Crossref PubMed Scopus (32) Google Scholar). It has been suggested more recently, based on the increased affinity of the two C-terminal Ca2+-binding sites of CaM in the presence of MLCK (Kd = 1.1 × 10−7m), that CaM at resting [Ca2+]i may contain two bound Ca2+ ions and therefore interacts with, but does not activate, MLCK (12Johnson J.D. Snyder C. Walsh M.P. Flynn M. J. Biol. Chem. 1996; 271: 761-767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). An increase in [Ca2+] in the vicinity of the myofilaments would then saturate the remaining two (the N-terminal) Ca2+-binding sites of CaM (Kd = 2.6 × 10−6m) (12Johnson J.D. Snyder C. Walsh M.P. Flynn M. J. Biol. Chem. 1996; 271: 761-767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), inducing a conformational change (collapse of CaM around the CaM-binding domain of MLCK) that leads to activation of the kinase (13Krueger J.K. Olah G.A. Rokop S.E. Zhi G. Stull J.T. Trewhella J. Biochemistry. 1997; 36: 6017-6023Crossref PubMed Scopus (63) Google Scholar,14Krueger J.K. Gallagher S.C. Zhi G. Geguchadze R. Persechini A. Stull J.T. Trewhella J. J. Biol. Chem. 2001; 276: 4535-4538Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). A number of observations suggest, however, that a pool of tightly bound CaM may be responsible for activating tension in smooth muscle (e.g. Refs. 15Filo R.S. Bohr D.F. Rüegg J.C. Science. 1965; 147: 1581-1583Crossref PubMed Scopus (180) Google Scholar, 16Gordon A.R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3527-3530Crossref PubMed Scopus (58) Google Scholar, 17Kerrick W.G.L. Hoar P.E. Cassidy P.S. Fed. Proc. 1980; 39: 1558-1563PubMed Google Scholar). Thus, skinned smooth muscle tissues retain the ability to contract in response to micromolar concentrations of Ca2+ and relax upon chelation of Ca2+, concomitant with LC20 phosphorylation and dephosphorylation, respectively. The addition of exogenous CaM to skinned smooth muscle preparations enhances the Ca2+sensitivity of contraction without affecting maximal tension development (e.g. Refs. 6Zimmermann B. Somlyo A.V. Ellis-Davies G.C.R. Kaplan J.H. Somlyo A.P. J. Biol. Chem. 1995; 270: 23966-23974Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar and 17Kerrick W.G.L. Hoar P.E. Cassidy P.S. Fed. Proc. 1980; 39: 1558-1563PubMed Google Scholar, 18Cassidy P.S. Kerrick W.G.L. Hoar P.E. Malencik D.A. Pflügers Arch. 1981; 392: 115-120Crossref PubMed Scopus (23) Google Scholar, 19Sparrow M.P. Mrwa U. Hofmann F. Rüegg J.C. FEBS Lett. 1981; 125: 141-145Crossref PubMed Scopus (101) Google Scholar, 20Rüegg J.C. Meisheri K. Pfitzer G. Zeugner C. Basic Res. Cardiol. 1983; 78: 462-471Crossref PubMed Scopus (21) Google Scholar). The total concentration of CaM in smooth muscle tissues is ∼10-fold higher than that of MLCK (∼40 μm (6Zimmermann B. Somlyo A.V. Ellis-Davies G.C.R. Kaplan J.H. Somlyo A.P. J. Biol. Chem. 1995; 270: 23966-23974Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 21Tansey M.G. Luby-Phelps K. Kamm K.E. Stull J.T. J. Biol. Chem. 1994; 269: 9912-9920Abstract Full Text PDF PubMed Google Scholar, 22Rüegg J.C. Pfitzer G. Zimmer M. Hofmann F. FEBS Lett. 1984; 170: 383-386Crossref PubMed Scopus (18) Google Scholar) versus∼3.4 μm (21Tansey M.G. Luby-Phelps K. Kamm K.E. Stull J.T. J. Biol. Chem. 1994; 269: 9912-9920Abstract Full Text PDF PubMed Google Scholar)), consistent with the fact that CaM associates with numerous proteins in addition to MLCK (23Vogel H.J. Biochem. Cell Biol. 1994; 72: 357-376Crossref PubMed Scopus (223) Google Scholar). In this study, we have tested the hypothesis that smooth muscle contraction involves the activation by Ca2+ of CaM that is tightly bound to the Triton X-100-insoluble fraction, even in the absence of Ca2+, and conclude that the current view of the mechanism of activation of smooth muscle contraction by Ca2+ and CaM requires substantial revision. Triton X-100 was purchased from Fisher, trifluoperazine (TFP) from ICN Biomedicals, Inc., CaM from Sigma, monoclonal anti-CaM from Upstate Biotechnology, Inc., anti-mouse IgG-horseradish peroxidase conjugate and anti-rabbit IgG-horseradish peroxidase conjugate from Roche Molecular Biochemicals, and 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine-HCl (ML-9) from Calbiochem-Novabiochem. HEPES-Tyrode (H-T) buffer contained 137.0 mm NaCl, 2.7 mm KCl, 1.0 mm MgCl2, 1.8 mm CaCl2, 10 mm HEPES, 5.6 mm glucose, pH 7.4. Ca2+-free H-T buffer contained 140.6 mm NaCl, 2.7 mm KCl, 1.0 mm MgCl2, 10 mm HEPES, 5.6 mm glucose, pH 7.4. Buffer A contained 30 mm TES, 0.5 mm dithiothreitol, 50 mm KCl, 5 mm K2EGTA, 150 mm sucrose, pH 7.4. pCa 9 solution contained 20 mm TES, 4 mm K2EGTA, 5.83 mm MgCl2, 7.56 mm potassium propionate, mm 0.5 mm mm pH The free [Ca2+] of this pCa 9 solution was to be solution contained 20 mm TES, 4 mm mm MgCl2, mm potassium propionate, mm mm 0.5 mm pH by and as by the of of and tissue free from the tail in Ca2+-free H-T tail a and and to the to and on a to a on the with a resting tension of and for 20 in H-T buffer = with H-T buffer mm (the increase in was by a in with a of in Ca2+-free H-T of and the is in Following a after the removal of muscle for 5 in Ca2+-free H-T buffer and for 5 in buffer A and skinned by for in buffer A Triton tissues for 5 in pCa 9 solution followed by for 30 solution to a Ca2+-induced contraction. muscle then by in pCa 9 solution for 10 and for × 5 in pCa 9 A Ca2+-induced contraction was as the of this contraction after the addition of was and the muscle to 30 tissues for 5 9 to pCa solution for 20 then to a contractile response. 9 solution for × 5 the solution was to CaM in pCa 9 solution 9 solution and unbound CaM was by for × 5 in pCa 9 then in pCa solution for 30 followed by pCa 9 solution for 5 of at as in for with monoclonal anti-CaM at or Walsh M.P. J. Physiol. 1999; Scopus Google Scholar) at a by in 10 mm on was by for in 10 in to for by for in of buffer Nature. PubMed Scopus Google Scholar). on and at a then for 10 in mm potassium pH and proteins to μm at for in mm potassium pH M. PubMed Scopus Google Scholar). are Walsh M.P. J. Physiol. 1999; Scopus Google Scholar). IgG-horseradish peroxidase conjugate and anti-rabbit IgG-horseradish peroxidase conjugate used as the at a of CaM it was to different of tissue protein to that the on the of the protein to be of The following used for this of a for and tissues and in for tissue and following the Ca2+-induced contraction 30 for other tissue and from tissue and and LC20 by to with as previously Walsh M.P. J. Physiol. 1999; Scopus Google Scholar). are as S.E. by A of was to be the number of a contraction of smooth muscle by induced a contraction, following which tension to a A to solution in The was then skinned by in solution Triton X-100 and 5 mm Following skinning in the absence of Ca2+, the retained the to contract in response to Ca2+ and upon the removal of Ca2+. The of the Ca2+-induced contraction was with that by of the tissue to A Ca2+-induced contraction was then at the of this contraction, mm was in the presence of Ca2+, in is a CaM antagonist that binds to CaM in the presence of Ca2+ and CaM from proteins B. Google Scholar) and has been to of skinned smooth muscle P. Hoar P.E. Kerrick W.G.L. Pflügers Arch. 1980; 115-120Crossref PubMed Scopus Google Scholar, W.G.L. Hoar P.E. Cassidy P.S. L. Malencik D.A. J. Physiol. 1981; PubMed Scopus Google Scholar). was then by in the absence of Ca2+. The addition of Ca2+ to a contractile response. Following in pCa 9 CaM was in the absence of Ca2+, and unbound CaM was then 9 The addition of in contraction that was on removal of Ca2+. a with an that CaM was not following In this the addition of to a contraction. data changes in tension different are in The CaM of muscle at different of the in by with anti-CaM In the in the of tissue was to to allow to be to of CaM in the different tissue it was to different of tissue protein to that the on the of the protein in as The CaM of muscle to and after the contraction and in was and as and was at was a in CaM following Triton skinning which was by the Ca2+-induced contraction with in the presence of Ca2+ and removal of the (Ca2+)4-CaM-TFP2 in of cellular CaM that this CaM was in the The of CaM in the absence of Ca2+ and washout of unbound CaM Ca2+-independent of exogenous CaM with and the which retained of total of the tissue with that the myosin was the The was for the protein not is required for removal of tightly bound The of the are as Following Ca2+-induced contraction of was induced by the removal of or the addition of in the presence of Ca2+ in the absence of Ca2+ for 5 in A or for 5 in followed by the addition of Ca2+ which induced a contractile response in A but not in B. C, the solution was after with in the presence of Ca2+ and The was in of buffer and of and 30 to and with The CaM is on the LC20 phosphorylation was also at different the of by and with A and data are in contraction of tissue with an increase in from an to of of to results with smooth muscle (e.g. Refs. P.E. Kerrick W.G.L. Cassidy P.S. Science. PubMed Scopus Google Scholar, J.T. M. K. J. Biol. Chem. 1980; Full Text PDF PubMed Google Scholar, Res. PubMed Scopus Google Scholar, M. Y. K. Walsh M.P. Biochem. J. 2000; PubMed Google Scholar). Following skinning in the absence of Ca2+ and removal of of cellular CaM, the addition of Ca2+ in an increase in from to of of 4 and The addition of at the of the Ca2+-induced contraction and LC20 Following removal of the (Ca2+)4-CaM-TFP2 complex, the addition of Ca2+ to increase consistent with to a contractile response The of CaM in the absence of Ca2+, followed by washout of unbound CaM, in the of a of CaM and restored Ca2+-dependent to of of to a contraction removal of Ca2+ in and of LC20 In tissues to which exogenous CaM was not the addition of Ca2+ to increase LC20 phosphorylation and to a contractile response The contractile response that was restored by of exogenous CaM in the absence of Ca2+ was by the M. S. M. Google Scholar) the for activation of by exogenous bound in the absence of Ca2+ is by as in the of The of the are not The Ca2+-induced contraction of muscle and to the removal of tightly bound CaM by with Following the removal of the (Ca2+)4-CaM-TFP2 complex, the addition of exogenous CaM in the absence of Ca2+, and washout of unbound CaM, muscle in the presence or absence of to the addition of Ca2+. was in and the concentration of in the solution was in both A and B. The Ca2+-induced contraction was by ML-9. are of = was required to the CaM that following skinning in the presence of in Ca2+-induced contraction of skinned RTA, followed by 9 not Ca2+-induced contraction, this contractile response as therefore, a pool of CaM, tightly bound to the Triton X-100-insoluble even in the absence of Ca2+, which is of an contractile response. The with anti-CaM in that CaM was by CaM also bound to at a [Ca2+] subthreshold for contraction to resting the that a of the exogenous CaM that binds to skinned smooth muscle in the absence of to a concentration of free CaM to for the of Ca2+-induced contraction. of the of CaM to the skinned muscle in the absence of Ca2+ we of this CaM released the solution the concentration of CaM would be this would be to Ca2+-dependent contraction of skinned RTA, we CaM with TFP, as Ca2+ which no and then CaM, in the presence of Ca2+. contraction no contraction even at or 20 CaM but the addition of a maximal contraction in the of the exogenous CaM, a contraction of maximal Ca2+-induced was observed of CaM to the muscle 20 also to a contraction and the of the addition of CaM to tissues at of the The contractile response to exogenous CaM bound in the absence of therefore, be to of this bound CaM the it Ca2+, to and with MLCK, and The fact that exogenous CaM bound in the absence of not to a was also by the that this CaM Ca2+-induced by washout in pCa 9 with anti-CaM that exogenous CaM bound in the absence of Ca2+ was retained following Ca2+ and washout not The results of this study that rat tail arterial smooth muscle a pool of CaM that is tightly bound to the at subthreshold [Ca2+]i, or even in the absence of Ca2+, and that the current view that to MLCK on the thin to activation is to be The of results is that CaM is to MLCK in the absence of Ca2+, since exogenous bound CaM a Ca2+-dependent contraction that is by the MLCK inhibitor ML-9, and is no for of the exogenous bound CaM from the suggest, therefore, that the contractile CaM is bound to a protein of the MLCK, although for be to this it that exogenous CaM is to a other than MLCK, this an since it would upon an increase in [Ca2+]i, the CaM would have to from this protein and to and MLCK, an the results in or the CaM would bound to this protein in the presence of Ca2+, would also be to to and MLCK. the CaM bound to MLCK in the absence of Ca2+, the of the kinase activated upon Ca2+ to CaM Walsh D.A. 1999; PubMed Google Scholar). the Ca2+-independent for CaM is MLCK, then the interaction CaM and the kinase be different from the interaction CaM and MLCK, which is Ca2+-dependent R.S. J. Biol. Chem. 1981; Full Text PDF PubMed Google Scholar, Stull J.T. J. Res. Cell 1997; PubMed Scopus Google Scholar). This the that a distinct protein of the myofilaments the the of MLCK bound to the myofilaments may be different from that of the free kinase in this be to CaM in a Ca2+-independent manner. therefore, that the pool of CaM that is involved in regulation of MLCK and smooth muscle contraction is tightly to the of troponin C with the myofilaments of skeletal and cardiac have suggested that of the CaM in a resting smooth muscle cell is unbound consistent with to a number of at the [Ca2+]i in the cell K. M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Persechini A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. B. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. C. J. Physiol. 2001; Google Scholar). of this bound CaM may be in with free cytosolic CaM, the is tightly as in the of the contractile CaM indicate tissue concentrations of μm for CaM (6Zimmermann B. Somlyo A.V. Ellis-Davies G.C.R. Kaplan J.H. Somlyo A.P. J. Biol. Chem. 1995; 270: 23966-23974Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 21Tansey M.G. Luby-Phelps K. Kamm K.E. Stull J.T. J. Biol. Chem. 1994; 269: 9912-9920Abstract Full Text PDF PubMed Google Scholar, 22Rüegg J.C. Pfitzer G. Zimmer M. Hofmann F. FEBS Lett. 1984; 170: 383-386Crossref PubMed Scopus (18) Google Scholar) and μm for MLCK (21Tansey M.G. Luby-Phelps K. Kamm K.E. Stull J.T. J. Biol. Chem. 1994; 269: 9912-9920Abstract Full Text PDF PubMed Google Scholar). data indicate that of total CaM binds to the after of CaM with and the addition of exogenous CaM in the absence of Ca2+, followed by washout of unbound CaM This to μm CaM to since of the MLCK is retained in the skinned smooth muscle Walsh M.P. J. Physiol. 1999; Scopus Google Scholar). This of bound CaM maximal Ca2+-induced contraction, since the addition of CaM at 9 of the in no development not results suggest, therefore, that the mechanism of activation of smooth muscle contraction by Ca2+ and CaM is as in as to the current in with observations with skinned smooth muscle this is to be a of smooth muscle (6Zimmermann B. Somlyo A.V. Ellis-Davies G.C.R. Kaplan J.H. Somlyo A.P. J. Biol. Chem. 1995; 270: 23966-23974Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Filo R.S. Bohr D.F. Rüegg J.C. Science. 1965; 147: 1581-1583Crossref PubMed Scopus (180) Google Scholar, 16Gordon A.R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3527-3530Crossref PubMed Scopus (58) Google Scholar, 17Kerrick W.G.L. Hoar P.E. Cassidy P.S. Fed. Proc. 1980; 39: 1558-1563PubMed Google Scholar, 18Cassidy P.S. Kerrick W.G.L. Hoar P.E. Malencik D.A. Pflügers Arch. 1981; 392: 115-120Crossref PubMed Scopus (23) Google Scholar, 19Sparrow M.P. Mrwa U. Hofmann F. Rüegg J.C. FEBS Lett. 1981; 125: 141-145Crossref PubMed Scopus (101) Google Scholar, 20Rüegg J.C. Meisheri K. Pfitzer G. Zeugner C. Basic Res. Cardiol. 1983; 78: 462-471Crossref PubMed Scopus (21) Google Scholar, 21Tansey M.G. Luby-Phelps K. Kamm K.E. Stull J.T. J. Biol. Chem. 1994; 269: 9912-9920Abstract Full Text PDF PubMed Google Scholar, 22Rüegg J.C. Pfitzer G. Zimmer M. Hofmann F. FEBS Lett. 1984; 170: 383-386Crossref PubMed Scopus (18) Google Scholar, P. Hoar P.E. Kerrick W.G.L. Pflügers Arch. 1980; 115-120Crossref PubMed Scopus Google Scholar, W.G.L. Hoar P.E. Cassidy P.S. L. Malencik D.A. J. Physiol. 1981; PubMed Scopus Google Scholar). of Ca2+ from the or from the is followed by to the it binds to CaM that is with MLCK to the thin and P. J. C. J. Physiol. 2001; Scopus Google Scholar) have a contractile Ca2+ is released from the the vicinity of the The of a pool of CaM bound to MLCK on the thin filament then be activated by this released Ca2+. then a distinct pool of Ca2+ the from the for that are in regions of the not to a regulate other Ca2+-dependent without a contractile response. data that CaM to MLCK in it is at resting [Ca2+]i D.A. J. Physiol. PubMed Google Scholar, D.A. Science. PubMed Scopus Google Scholar), at of the bound CaM two Ca2+ ions bound to the two C-terminal Ca2+-binding since the Ca2+ affinity of sites is to 1.1 × in the presence of a to the CaM-binding domain of MLCK (12Johnson J.D. Snyder C. Walsh M.P. Flynn M. J. Biol. Chem. 1996; 271: 761-767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). results that the of CaM to MLCK in the tissue does not bound Ca2+. resting [Ca2+]i, therefore, contractile CaM is bound to MLCK and of a of and of which is of activation of the the [Ca2+] in the vicinity of the myofilaments a Ca2+ saturate the four Ca2+-binding sites of CaM, inducing a conformational change that is to MLCK (13Krueger J.K. Olah G.A. Rokop S.E. Zhi G. Stull J.T. Trewhella J. Biochemistry. 1997; 36: 6017-6023Crossref PubMed Scopus (63) Google Scholar), in its the of the Ca2+-binding sites is 2.6 × (12Johnson J.D. Snyder C. Walsh M.P. Flynn M. J. Biol. Chem. 1996; 271: 761-767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). MLCK then the of the myosin triggering cross-bridge cycling. This mechanism of resembles that of striated with CaM to the Ca2+-binding protein troponin C, which is also bound to the thin the of troponin C with the myofilaments is by this does not to be the for CaM, which binds Ca2+ at concentrations of Ca2+, and Chem. 1982; PubMed Scopus Google Scholar). the mechanism in has the the for activation of smooth muscle contraction it local changes in [Ca2+] from the contractile to regulate distinct (e.g. regulation of or without a contractile response. the mechanism of activation of as cell and contraction, is also to phosphorylation of myosin K.E. Stull J.T. J. Biol. Chem. 2001; 276: 4527-4530Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), this is also of to regulation of are to and of J. Hartshorne of Stull of and of for and on the The of the results is the of the

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