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  1. 920 Biochemical Society Transactions (2003) Volume 31, part 5 Sarcolemma agonist-induced interactions between InsP3and ryanodine receptors in Ca2+ oscillations and waves in smooth muscle J.G. McCarron1, K.N. Bradley, D. MacMillan and T.C. Muir Institute of Biomedical and Life Sciences, Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, U.K. Abstract Smooth muscle cells respond to InsP3-generating (sarcolemma-acting) neurotransmitters and hormones by releasing Ca2+from the internal store. However, the release of Ca2+does not occur uniformly throughout the cytoplasm but often into a localized area before being transmitted to other regions of the cell in the form of Ca2+waves and oscillations to actively spread information within and between cells. Yet, despite their significance, our understanding of the generation of oscillations to waves is incomplete. A major aspect of controversy centres on whether or not Ca2+released from the InsP3receptor activates RyRs (ryanodine receptors) to generate further release by Ca2+-induced Ca2+release and propagate waves or whether the entire process arises from InsP3receptor activity alone. Under normal physiological conditions the [Ca2+] required to activate RyR (approx. 15 µM) exceeds the bulk average [Ca2+]c(cytoplasmic Ca2+concentration) generated by InsP3receptor activity (<1 µM). Progression of waves and oscillations by RyR activity would require a loss of control of RyR activity and an unrestrained positive feedback on Ca2+release. Under store-overload conditions, RyR Ca2+sensitivity is increased and this enables waves to be induced by RyR activity. However, the relevance of these Ca2+-release events to normal physiological functioning is unclear. The InsP3receptor, on the other hand, is activated by Ca2+over the physiological range (up to 300 nM) and deactivated by higher [Ca2+]c (>300 nM), features that favour intermittent activity of the receptor as occurs in waves and oscillations. Experimental evidence for the involvement of RyR relies mainly on pharmacological approaches in the intact cell where poor drug specificity could have led to ambiguous results. In this brief review the possible interactions between InsP3receptors and RyR in the generation of oscillations and waves will be discussed. Evidence is presented that RyRs are not required for InsP3-mediated Ca2+transients. Notwithstanding, ryanodine can inhibit InsP3-mediated Ca2+responses after RyR activity has been induced by caffeine or by steady depolarization which evokes spontaneous transient outward currents (a sarcolemmal manifestation of RyR activity). Ryanodine inhibits InsP3-mediated Ca2+transients by depleting the store of Ca2+rather than by RyR involvement in the InsP3-mediated Ca2+increase. Downloaded from by guest on 04 May 2020 Introduction Smooth muscle resting [Ca2+]c(cytoplasmic Ca2+concen- tration) is approx. 100 nM [1,2]; in response to excitatory transmitters which generate InsP3, this increases by several hundredfold in a few seconds [3]. The initial rise in [Ca2+]cin responseto InsP3-generating transmittershas been attributed, traditionally, to a large transient release from the SR (sarcoplasmic reticulum), followed by a secondary, lower sustained increase in [Ca2+]cto above resting levels, the latter depending on extracellular Ca2+influx through sarcolemmal Ca2+channels [4]. However, re-examination of the changes in [Ca2+]cin single cells within tissues has shown that each cell does not respond to stimulation with a co-ordinated transient and a sustained increase in [Ca2+]c[5–8]. Rather, at the subcellular level, Ca2+signals appear as localized events – Ca2+‘puffs’ and ‘sparks’ – which are restricted to a small area ofthecellandwhichcomprisetheelementarycomponentsof the global Ca2+response [9,10]. Under certain, as-yet- unspecifiedconditions,Ca2+puffsand/orsparksmayinteract with each other so that the cytoplasm becomes ‘excitable’ andlocalevents coalescetobecomerepetitiveCa2+transients called oscillations. These oscillations may, in turn, propagate as Ca2+waves and spread information within cells and be- tween cells [11,12]. At the tissue level, the oscillations and waves summate to generate the traditionally described bi- phasic changes in [Ca2+]c, i.e. the initial large Ca2+transient followed by a second lower sustained increase in [Ca2+]c(e.g. [6,13–15]). Several advantages to the Ca2+signalling process would seem to accrue from the cell’s ability to produce oscillations and waves. For example, several enzymes (e.g. calmodulin kinase II and protein kinase C [16,17]) are activated at the frequenciesoftheseCa2+oscillations,inpreferencetosteady- state [Ca2+]crises. The maintenance of a steady, high [Ca2+]c, on the other hand, may be apoptopic. Key words: Ca2+imaging, Ca2+signalling, InsP3receptor, ryanodine receptor, voltage clamp. Abbreviations used: [Ca2+]c, cytoplasmic Ca2+concentration; SR, sarcoplasmic reticulum; RyR, ryanodine receptor; CICR, Ca2+-induced Ca2+release. 1To whom correspondence should be addressed (e-mail C ?2003 Biochemical Society

  2. Calcium Oscillations and the 5th UK Calcium Signalling Conference 921 Ca2+oscillations contribute to the selective activation of transcriptional factors and gene expression [18,19] in Jurkat T-cells, events likely to recur in smooth muscle [20]. Yet, despite their significance in Ca2+signalling, the underlying events which trigger oscillations and govern the propagation of Ca2+waves are unclear. Several proposals have been made; nonehasgaineduniversalacceptance.Ofthese,twohavebeen prominent and involve those receptor complexes on the SR which govern Ca2+release, i.e. the InsP3receptor and RyR (ryanodine receptor). sients, negative feedback mechanisms may also control transient frequency. The interval between oscillations may reflect the time required by the InsP3 receptor to recover from the feedback deactivation [35,37,38]. Interactions between RyR and InsP3 receptors in the production of oscillations and waves It is important to evaluate the precise role of each receptor complex in order to understand the origin of Ca2+waves. Most investigations of this problem have relied heavily on pharmacological approaches, e.g. using the RyR inhibitors ryanodine and Ruthenium Red. In the absence of precise information on the state of filling of the store, the complex action of ryanodine makes interpretation of its effects on InsP3-mediated responses difficult. Similarly, drugs like Ruthenium Red are also known in some cell types to have complex actions on InsP3-mediated Ca2+responses. For example, in avian atria Ruthenium Red inhibited the response to InsP3 by an action unrelated to RyR since the response to caffeine was potentiated [39]. Nonetheless a considerable body of potential support for RyR involvement in oscillations and waves in smooth muscle exists. For example, in porcine tracheal smooth muscle cells ryanodine and Ruthenium Red inhibited acetylcholine-induced Ca2+ oscillations[40,41].TheconclusionwasthatRyRcontributed to the acetylcholine-induced Ca2+increase. Similarly, the P2Y (metabotropic purinergic) receptor agonists uridine 5?- triphosphate and 2-methylthio-ATP each increased the oc- currence of Ca2+waves in rat cerebral artery and murine colonic smooth muscle cells; each was blocked by ryanodine [42,43]. In rabbit cerebral artery, histamine-induced Ca2+ oscillationswereblockedbyryanodine;theauthorssuggested that RyR-dependent CICR contributed to the Ca2+rise [44]. In rabbit portal vein myocytes Ca2+release via the InsP3 receptor reportedly may activate CICR from neighbouring RyR [45]. In rat portal vein smooth muscle cells, noradrenaline-induced Ca2+release was reduced by the RyR blocker ryanodine or by a ryanodine receptor antibody; InsP3-mediated Ca2+release in this study, it was suggested, was amplified by RyR activity [22,46]. However, in another study also on rat portal vein myocytes, although the RyR blocker tetracaine inhibited Ca2+release evoked by the RyR activator caffeine (a control experiment), the blocker did not reduce the Ca2+increase initiated by the InsP3-generating sarcolemma agonist noradrenaline. These results suggest that InsP3-mediated Ca2+release in rat portal vein myocytes is not amplified by CICR acting at RyR [47]. Perhaps these contradictory results obtained on the same smooth muscle preparation, from the same species, could be explained by the absence of both ATP and magnesium from the intracellular solution in the former studies [22,46]. Relieving the inhibitory effect of magnesium on Ca2+release and reducing phosphorylation of RyR could have increased the channel sensitivity to Ca2+liberated via InsP3receptors. Alternatively, the culture conditions in which the cells were Origin of Ca2+oscillations and waves The two SR receptor/channel complexes are activated by either InsP3(the InsP3receptor) or by CICR (Ca2+-induced Ca2+release via RyR) and oscillations and wave propagation may result from an interplay between them [21]. There is generalagreementthattheinitiationofoscillationsandwaves is a response to sarcolemma-acting agonists which release Ca2+from the SR via InsP3 receptors [21]. Controversy persists, however, concerning the subsequent propagation of the wave. The major aspect of this controversy centres on whether or not Ca2+released from the InsP3 receptor then activates RyR to generate further release by CICR and to propagate waves, or whether the entire release process arises from InsP3receptor activity alone without significant RyR involvement. Thus it may be that the initiating InsP3- mediated Ca2+release is followed by a more substantial Ca2+release from the RyR via CICR which in turn activates more RyR nearby so that a cycle of release and diffusion of Ca2+throughout the cell ensues. In support of this proposal, drugs which alter RyR conductance, such as ryanodine and Ruthenium Red, often, but not invariably, abolished [Ca2+]c oscillations (e.g. [13,15,22,23]; see below). Yet in some cell types which do not have a caffeine- sensitive CICR mechanism, Ca2+waves are evident [24,25]. Another proposal for the periodicity of Ca2+oscillations, therefore, relies more exclusively on feedback mechanisms at the InsP3 receptor between Ca2+and InsP3 [26–28]. In this, oscillations, it is proposed, originate from properties inherent in the InsP3receptors themselves which allow them to open and close (deactivate) in the continued presence of a constant concentration of InsP3(e.g. [29,30]). This proposal requires that (i) a synergistic action exists between Ca2+ and InsP3in which the InsP3receptor may act as a CICR site, (ii) a high [Ca2+]cinhibits InsP3receptor opening and (iii) a refractory period for the InsP3-gated channel follows channel opening; this period is induced by InsP3itself or by cytoplasmic and/or luminal [Ca2+] [31–36]. Thus the rapid upstroke of the Ca2+transient is mediated by activation of a positive feedback mechanism or a sensitization of the InsP3 receptor by released Ca2+. The subsequent termination of release and the fall of [Ca2+]c to basal levels follow from a feedback deactivation of the InsP3 receptor. Indeed, an intrinsicfeedback deactivation of the InsP3receptor may also contribute to enable the SR pump to restore SR Ca2+levels [35].Aswellascausingthefallingphaseoftheindividualtran- Downloaded from by guest on 04 May 2020 C ?2003 Biochemical Society

  3. 922 Biochemical Society Transactions (2003) Volume 31, part 5 maintained[22]mayhavecausedphenotypicchanges.Indeed in the cultured vascular cell line A7r5, vasopressin-induced Ca2+waves were propagated by CICR acting on RyR [48]. Results from experiments examining their participation in InsP3-mediated Ca2+release fall short of incontrovertible support for RyR involvement. Indeed a considerable number of experiments found little support for such a role for RyR in this process and proposed that InsP3 receptors alone were responsible. For example, in equine tracheal myocytes, the Ca2+release evoked by the InsP3-generating muscarinic agonist methacholine was unaffected while that evoked by caffeine (as a control) was blocked by the RyR blocker Ruthenium Red [49]. In rat ureteric myocytes the Ca2+waves evoked by photolysis of caged InsP3 were blocked by InsP3 receptor inhibitors such as heparin but unaffectedbyRyRinhibitionwithryanodine[50].Ryanodine blocked the caffeine responses of the guinea pig ureter but not those to carbachol in rat ureter, suggesting that carbachol was releasing Ca2+from a ryanodine-insensitive channel [51,52]. In guinea pig intestinal smooth muscle, Ca2+ oscillations evoked by muscarinic receptor activation were unaffectedbyRutheniumRed[53].Inratpulmonaryarteries, endothelin-evoked Ca2+oscillations also occurred as a result of Ca2+release from the internal store. The RyR inhibitors Ruthenium Red and tetracaine (each of which abolished the Ca2+increase evoked by caffeine) did not affect the Ca2+ oscillations [23]. In guinea pig colonic myocytes repetitive Ca2+-release events from the InsP3receptor by photolysis of caged InsP3were not inhibited by either tetracaine [54] or ryanodine [55] in the absence of prior RyR activity; InsP3receptor activity alone accounted for the Ca2+increase. Noradrenaline-evoked Ca2+waves persisted in rat tail artery segments maintained in organ culture for days with a non- deactivating ryanodine analogue, while Ca2+release evoked by caffeine was lost. These results suggest that RyR does not contribute to waves [56]. Likewise RyR played little role in amplifying InsP3-mediated Ca2+increases in guinea pig intestinal smooth muscle [57]. Together these results point to the InsP3receptor alone accounting for Ca2+waves and oscillations. In addition to the difficulties associated with the multi- plicity of RyR-blocking drugs’ actions, e.g. Ruthenium Red, the apparent contradictory evidence from experiments which examined the roles of RyR and InsP3 receptors in wave propagation may derive in part from two additional sources (a) differences in the interpretation of the results and (b) the creation by the experimental protocol of abnormal store- overload conditions in which the sensitivity of the receptors to Ca2+may have been changed. Ryanodine and Ruthenium Red, for example, may each inhibit InsP3-mediated Ca2+ signalsindependentlyoftheinvolvementofRyRintheInsP3- mediated response [13,54,55,58,59]. In the case of ryanodine this is due to its ability to deplete SR Ca2+stores to which InsP3receptors and RyR have common access. For example, acetylcholine, noradrenaline and nerve stimulation each released Ca2+in porcine coronary, rabbit mesenteric and rat tail arteries respectively [13,60,61]. The release of Ca2+by the neurotransmitters was inhibited by ryanodine in each study and attributed to the drug’s ability to open RyRs and deplete the store of Ca2+[13,60,61]. In our studies on colonic myocytes in which ryanodine blocked InsP3-mediated Ca2+ increases, the results were attributed to the drug’s indirect inhibition of the InsP3-mediated response by store depletion of Ca2+rather than to involvement of RyR in Ca2+increase [55].Forexample,ryanodine(50 µM)byitselfdidnotinhibit InsP3-evoked Ca2+increases (Figure 1). RyR activation with caffeine evoked a substantial increase in [Ca2+]c. The second caffeine application in the same cell was inhibited. Thereafter the response to InsP3 was lost (Figure 1). These results suggest that InsP3-mediated Ca2+release does not activate RyR. Notwithstanding, ryanodine can block InsP3-mediated Ca2+transients. Thus after RyR activity was induced (by caffeine) ryanodine presumably locked RyRs open [62] and depleted the store of Ca2+, conditions resulting in the loss of InsP3-mediated Ca2+transients (Figure 1; see also [55]). Indeed, it is also possible for ryanodine to inhibit InsP3- evoked Ca2+transients without prior caffeine exposure but still without the involvement of RyR in the InsP3response. Thus at negative membrane potentials, at which RyR is relatively inactive [63], ryanodine did not reduce the InsP3- mediated Ca2+transient [55]. However, at –20 mV, at which RyRs are active and generating spontaneous transient outward currents, ryanodine could inhibit InsP3-mediated Ca2+increases [54]. The latter result occurred because of partial depletion of the store by ryanodine locking RyRs in the open state rather than by the involvement of RyRs in the InsP3response, since tetracaine had no effect on the InsP3- mediatedCa2+responseatthesamemembranepotential[54]. If RyRs are involved in InsP3-mediated responses the normal control mechanisms for Ca2+release from the store would need to be compromised. Under physiological con- ditions, the [Ca2+]c required to activate RyRs is approx. 15 µM [64] in heart cells at least. The InsP3 receptor, on the other hand, is deactivated by Ca2+concentrations which exceed 300 nM [26–28]. The low Ca2+sensitivity of RyR contributes to ‘local control’ [65] so that Ca2+release does notactivateneighbouringRyRsandcanbecontrolled,graded and terminated [66,67]. Implicit in this view of wave pro- gression, by RyR activity, is that local control is lost and that uncontrolled positive feedback of Ca2+release occurs. Such uncontrolled release occurs when the SR contains excessively high levels of Ca2+– as in store overload. Here RyR sensitivity to Ca2+is increased so that the release of the ion occurs at lower [Ca2+]c. Indeed, in cardiac myocytes [68– 72], RyR-mediated Ca2+waves have been observed in store- overload conditions although the relevance of this to normal physiological functioning is unclear. It is tempting to suggest thatstore-overloadconditionswhichincreaseRyRsensitivity to Ca2+may promote activity of the channel at [Ca2+]c generated by InsP3-mediated Ca2+release. In response to InsP3-generating (sarcolemma-acting) agonists, InsP3 receptor activity alone may explain wave propagation in smooth muscle under physiological condi- tions but a direct demonstration of this is awaited. Our Downloaded from by guest on 04 May 2020 C ?2003 Biochemical Society

  4. Calcium Oscillations and the 5th UK Calcium Signalling Conference 923 Effect of ryanodine on InsP3- and caffeine-evoked Ca2+increases Photolysis of caged InsP3(↑) evoked approximately reproducible increases in [Ca2+]cin a single voltage-clamped colonic myocyte (A); ryanodine (50 µM) did not significantly reduce the InsP3-evoked Ca2+transients. (B) Activation of RyRs by caffeine(10 mM),inthecontinuedpresenceofryanodine,increased[Ca2+]c.Asecondapplicationofcaffeinetothesamecell some 80 s later generated little increase in [Ca2+]c, presumably because the SR store had been depleted of Ca2+. The InsP3 response was subsequently inhibited (↑; A). Because the InsP3-evoked Ca2+transient was not blocked by ryanodine alone (only after RyR activation with caffeine), InsP3-mediated Ca2+release does not activate RyRs. InsP3receptors and RyRs may share a common Ca2+store; this is depleted of Ca2+by ryanodine and caffeine. Figure 1 Downloaded from by guest on 04 May 2020 present investigations have centred on the mechanisms underlying InsP3-mediated Ca2+waves in smooth muscle either by increasing InsP3throughout the cell or restricting itsapplicationtosmalldiscreteregions(bylocalizeduncaging of InsP3). Initialfindingssuggest that whileincreases inInsP3 throughout the cell evoke Ca2+waves, localized increases in InsP3cannot – even when RyRs were active. RyR activity is not required for wave production; InsP3receptor activity alone is sufficient. 14 Zang, W.-J., Balke, C.W. and Wier, W.G. (2001) Cell Calcium 29, 327–334 15 Ruehlmann, D.O., Lee, C.-H., Poburko, D. and vanBreemen, C. (2002) Circ. Res. 86, e72–e79 16 DeKoninick, P. and Schulman, H. (1998) Science 279, 227–230 17 Oancea, E. and Meyer, T. (1998) Cell 95, 307–318 18 Dolmetsch, R.E., Xu, K. and Lewis, R.S. (1998) Nature (London) 392, 933–936 19 Li, W.-H., Llopis, J., Whitney, M., Zlokarnik, G. and Tsien, R.Y. (1998) Nature (London) 392, 936–941 20 Cartin, L., Lounsbury, K.M. and Nelson, M.T. (2000) Circ. Res. 86, 760–767 21 Goldbeter, A., Dumont, G. and Berridge, M.J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1461–1465 22 Boittin, F.-X., Macrez, N., Halet, G. and Mironneau, J. (1999) Am. J. Physiol. 277, C139–C151 23 Hyvelin, J.-M., Guibert, C., Marthan, R. and Savineau, J.-P. (1998) Am. J. Physiol. 275, L269–L282 24 DeLisle, S. and Welsh, M.J. (1992) J. Biol. Chem. 267, 7963–7966 25 Lechleiter, J.D. and Clapham, D.E. (1992) Cell 69, 283–294 26 Iino, M. (1990) J. Gen. Physiol. 95, 1103–1122 27 Bezprozvanny, I., Watras, J. and Ehrlich, B.E. (1991) Nature (London) 351, 751–754 28 Finch, E.A., Turner, T.J. and Goldin, S.M. (1991) Science 252, 443–446 29 Hajnoczky, G. and Thomas, A.P. (1997) EMBO J. 16, 3533–3543 30 Wakui, M., Potter, B.V.L. and Petersen, O.H. (1989) Nature (London) 339, 317–320 31 Hajnoczky, G. and Thomas, A.P. (1994) Nature (London) 370, 474–477 32 Marshall, I.C.B. and Taylor, C.W. (1993) J. Biol. Chem. 268, 13214–13220 33 Missiaen, L., DeSmedt, H., Droogmans, G. and Casteels, R. (1992) Nature (London) 357, 599–602 34 Nunn, D.L. and Taylor, C.W. (1992) Mol. Pharmacol. 41, 115–119 35 Oancea, E. and Meyer, T. (1996) J. Biol. Chem. 271, 17253–17260 36 Parker, I. and Ivorra, I. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 260–264 37 Atri, A., Amundson, J., Clapham, D. and Sneyd, J. (1993) Biophys. J. 65, 1727–1739 38 DeYoung, G.W. and Keizer, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9895–9899 39 Vites, A.M. and Pappano, A.J. (1992) Am. J. Physiol. 262, H268–H277 40 Kannan, M.S., Prakash, Y.S., Brenner, T., Mickelson, J.R. and Sieck, G.C. (1997) Am. J. Physiol. 272, L659–L664 The Wellcome Trust (060094/Z/00/Z) and the British Heart Foundation (PG/2001079; PG/02/161/14788) funded this work. References 1 Neering, I.R. and Morgan, K.G. (1980) Nature (London) 288, 585–587 2 Williams, D.A., Fogarty, R.Y., Tsien, R.Y. and Fay, F.S. (1985) Nature (London) 318, 558–561 3 Morgan, J.P. and Morgan, K.G. (1984) J. Physiol. 351, 155–167 4 Williams, D.A. and Fay, F.S. (1986) Am. J. Physiol. 250, C779–C791 5 Mahoney, M.G., Slakey, L.L., Hepler, P.K. and Gross, D.J. (1993) J. Cell Sci. 104, 1101–1107 6 Mauban, J.R.H., Lamont, C., Balke, C.W. and Wier, W.G. (2001) Am. J. Physiol. 280, H2399–H2405 7 Miriel, V.A., Mauban, J.R.H., Blaustein, M.P. and Wier, W.G. (1999) J. Physiol. 518, 815–825 8 Asada, Y., Yamazawa, T., Hirose, K., Takasaka, T. and Iino, M. (1999) J. Physiol. 521, 497–505 9 Bootman, M.D., Berridge, M.J. and Lipp, P. (1997) Cell 91, 367–373 10 Berridge, M.J., Lipp, P. and Bootman, M.D. (2000) Mol. Cell Biol. 1, 11–21 11 Lansley, A.B. and Sanderson, M.J. (1999) Biophys. J. 77, 629–638 12 Robb-Gaspers, L.D. and Thomas, A.P. (1995) J. Biol. Chem. 270, 8102–8107 13 Iino, M., Kasai, H. and Yamazawa, T. (1994) EMBO J. 13, 5026–5031 C ?2003 Biochemical Society

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