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• W1967440360 abstract "Myotubes expressing wild type RyR1 (WT) or RyR1 with one of three malignant hyperthermia mutations R615C, R2163C, and T4826I (MH) were exposed sequentially to 60 mm KCl in Ca2+-replete and Ca2+-free external buffers (Ca+ and Ca–, respectively) with 3 min of rest between exposures. Although the maximal peak amplitude of the Ca2+ transients during K+ depolarization was similar for WT and MH in both external buffers, the rate of decay of the sustained phase of the transient during K+ depolarization (decay rate) in Ca+ was 50% slower for MH. This difference was eliminated in Ca–, and the relative decay rates were faster for both genotypes than in Ca+. The integrated Ca2+ transient in Ca–compared with Ca+ was reduced by 50–60% for MH and 20% for WT. The decay rate was not affected by [K+] × [Cl–] product or NiCl2 (2 mm) supplementation of Ca–. The addition of La2+ (0.1 mm), or SKF 96365 (20 μm) to Ca+ significantly accelerated decay rates for both WT and MH, but their effect was significantly greater in MH. Nifedipine (1 μm) had no effect, suggesting that the mechanism for this difference was not a reduction in L-type Ca2+ channel Ca2+ current. These data strongly suggest: 1) the decay rate in skeletal myotubes is related in part to Ca2+ entry through the ECCE channel; 2) the MH mutations enhance ECCE compared with wild type; and 3) the increased Ca2+ entry might play a significant role in the pathophysiology of MH. Myotubes expressing wild type RyR1 (WT) or RyR1 with one of three malignant hyperthermia mutations R615C, R2163C, and T4826I (MH) were exposed sequentially to 60 mm KCl in Ca2+-replete and Ca2+-free external buffers (Ca+ and Ca–, respectively) with 3 min of rest between exposures. Although the maximal peak amplitude of the Ca2+ transients during K+ depolarization was similar for WT and MH in both external buffers, the rate of decay of the sustained phase of the transient during K+ depolarization (decay rate) in Ca+ was 50% slower for MH. This difference was eliminated in Ca–, and the relative decay rates were faster for both genotypes than in Ca+. The integrated Ca2+ transient in Ca–compared with Ca+ was reduced by 50–60% for MH and 20% for WT. The decay rate was not affected by [K+] × [Cl–] product or NiCl2 (2 mm) supplementation of Ca–. The addition of La2+ (0.1 mm), or SKF 96365 (20 μm) to Ca+ significantly accelerated decay rates for both WT and MH, but their effect was significantly greater in MH. Nifedipine (1 μm) had no effect, suggesting that the mechanism for this difference was not a reduction in L-type Ca2+ channel Ca2+ current. These data strongly suggest: 1) the decay rate in skeletal myotubes is related in part to Ca2+ entry through the ECCE channel; 2) the MH mutations enhance ECCE compared with wild type; and 3) the increased Ca2+ entry might play a significant role in the pathophysiology of MH. Excitation-contraction (EC) coupling 2The abbreviations used are: ECexcitation-contractionWTwild typeMHmalignant hyperthermiaDHPRdihydropyridine receptorSRsarcoplasmic reticulumSOCEstore-operated Ca2+ entryECCEexcitation-coupled Ca2+ entryIBimaging buffer 2The abbreviations used are: ECexcitation-contractionWTwild typeMHmalignant hyperthermiaDHPRdihydropyridine receptorSRsarcoplasmic reticulumSOCEstore-operated Ca2+ entryECCEexcitation-coupled Ca2+ entryIBimaging buffer in skeletal muscle is a cascade of events that is initiated by the depolarization of T-tubule membrane (dihydropyridine receptor (DHPR)), which is followed by activation of the intracellular Ca2+ release channels known as ryanodine receptors (RyR1) located at the terminal cisternae of the sarcoplasmic reticulum (SR). These two proteins align during development at the junction of the t-tubule with the terminal cisternae of the SR (1Flucher B.E. Franzini-Armstrong C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8101-8106Crossref PubMed Scopus (188) Google Scholar), a collection of structures termed the calcium release unit (2Protasi F. Franzini-Armstrong C. Allen P.D. J. Cell Biol. 1998; 140: 831-842Crossref PubMed Scopus (114) Google Scholar). The DHPR may serve a dual function as a slow activated voltage-dependent Ca2+ channel (3Rios E. Pizarro G. Physiol. Rev. 1991; 71: 849-908Crossref PubMed Scopus (495) Google Scholar) and as a voltage sensor for EC coupling (4Rios E. Brum G. Nature. 1987; 325: 717-720Crossref PubMed Scopus (650) Google Scholar). The DHPR is responsible for activating RyR1 causing Ca2+ release from the SR (5Schneider M.F. Chandler W.K. Nature. 1973; 242: 244-246Crossref PubMed Scopus (650) Google Scholar, 6Marty I. Robert M. Villaz M. De Jongh K. Lai Y. Catterall W.A. Ronjat M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2270-2274Crossref PubMed Scopus (139) Google Scholar, 7Beam K.G. Tanabe T. Numa S. Ann. N. Y. Acad. Sci. 1989; 560: 127-137Crossref PubMed Scopus (23) Google Scholar) into the cytoplasm that in turn triggers muscle contraction. Although the exact signal transduction mechanisms between DHPR and RyR1 are still unknown, it is generally accepted that intramembrane charge movements and conformational changes in the DHPR II-III loop couple the T tubule depolarization and Ca2+ release from the SR (orthograde conformational coupling) (5Schneider M.F. Chandler W.K. Nature. 1973; 242: 244-246Crossref PubMed Scopus (650) Google Scholar, 8Tanabe T. Mikami A. Niidome T. Numa S. Adams B.A. Beam K.G. Ann. N. Y. Acad. Sci. 1993; 707: 81-86Crossref PubMed Scopus (23) Google Scholar, 9Grabner M. Dirksen R.T. Suda N. Beam K.G. J. Biol. Chem. 1999; 274: 21913-21919Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). It is this massive release of Ca2+ from the SR into the cytosol, and not a Ca2+ influx from the extracellular space, that is conventionally thought to initiate a series of Ca2+-dependent events that results in force generation (10Anderson K. Cohn A.H. Meissner G. Am. J. Physiol. 1994; 266: C462-C466Crossref PubMed Google Scholar, 11Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (592) Google Scholar). excitation-contraction wild type malignant hyperthermia dihydropyridine receptor sarcoplasmic reticulum store-operated Ca2+ entry excitation-coupled Ca2+ entry imaging buffer excitation-contraction wild type malignant hyperthermia dihydropyridine receptor sarcoplasmic reticulum store-operated Ca2+ entry excitation-coupled Ca2+ entry imaging buffer Experiments in which external Ca2+ was removed (12Caputo C. Gimenez M. J. Gen. Physiol. 1967; 50: 2177-2195Crossref PubMed Scopus (45) Google Scholar, 13Armstrong C.M. Bezanilla F.M. Horowicz P. Biochim. Biophys. Acta. 1972; 267: 605-608Crossref PubMed Scopus (298) Google Scholar) or calcium channel blockers are added to the bathing medium around skeletal muscle fibers (14Gonzalez-Serratos H. Valle-Aguilera R. Lathrop D.A. Garcia M.C. Nature. 1982; 298: 292-294Crossref PubMed Scopus (100) Google Scholar) show that EC coupling and twitch contraction persists in skeletal muscle cells under these conditions. However, the role of extracellular Ca2+ on EC coupling in mammalian skeletal muscle has been re-examined (15Berridge M.J. Bioessays. 1995; 17: 491-500Crossref PubMed Scopus (456) Google Scholar). Several groups have demonstrated that stimuli that deplete Ca2+ in the SR enhance Ca2+ entry through the plasma membrane by a mechanism referred as store-operated Ca2+ entry (SOCE) (16Kurebayashi N. Ogawa Y. J. Physiol. 2001; 533: 185-199Crossref PubMed Scopus (237) Google Scholar, 17Ma J. Pan Z. Cell Calcium. 2003; 33: 375-384Crossref PubMed Scopus (51) Google Scholar). Although Ca2+ entry through SOCE is easily seen, identification of the channel(s) responsible still awaits discovery. It is highly likely that the mechanism for this entry is at least partially if not completely derived from current passing though Orai 1, 2, and/or 3, which is activated by translocation of Stim 1 or Stim 2 from the SR (18Huang G.N. Zeng W. Kim J.Y. Yuan J.P. Han L. Muallem S. Worley P.F. Nat. Cell Biol. 2006; 8: 1003-1010Crossref PubMed Scopus (566) Google Scholar, 19Luik R.M. Wu M.M. Buchanan J. Lewis R.S. J. Cell Biol. 2006; 174: 815-825Crossref PubMed Scopus (537) Google Scholar, 20Ong H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D. Ambudkar I.S. J. Biol. Chem. 2007; 282 (Correction (2007) J. Biol. Chem. 282, 27556): 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 21Prakriya M. Feske S. Gwack Y. Srikanth S. Rao A. Hogan P.G. Nature. 2006; 443: 230-233Crossref PubMed Scopus (1110) Google Scholar, 22Robinson L.C. Marchant J.S. Curr. Biol. 2006; 16: R548-R550Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 23Soboloff J. Spassova M.A. Tang X.D. Hewavitharana T. Xu W. Gill D.L. J. Biol. Chem. 2006; 281: 20661-20665Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). In addition to SOCE, a new mechanism for Ca2+ entry has recently been described in skeletal myotubes (24Cherednichenko G. Hurne A.M. Fessenden J.D. Lee E.H. Allen P.D. Beam K.G. Pessah I.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15793-15798Crossref PubMed Scopus (99) Google Scholar, 25Hurne A.M. O'Brien J.J. Wingrove D. Cherednichenko G. Allen P.D. Beam K.G. Pessah I.N. J. Biol. Chem. 2005; 280: 36994-37004Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) that is not linked to Ca2+ depletion of the SR and is elicited by brief or sustained membrane depolarization that would block conventional SOCE current(s). Activation of this Ca2+ entry pathway depends on interaction among three different Ca2+ channels: the DHPR, RyR1, and an unidentified Ca2+ influx pathway through the plasma membrane that was termed excitation-coupled Ca2+ entry (ECCE) (24Cherednichenko G. Hurne A.M. Fessenden J.D. Lee E.H. Allen P.D. Beam K.G. Pessah I.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15793-15798Crossref PubMed Scopus (99) Google Scholar). Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome in which exposure to volatile anesthetics or depolarizing neuromuscular blockers triggers a robust intracellular Ca2+ release through RyR1 from the SR, inducing a cascade of biochemical events that if untreated results in muscle rigidity, rhabdomyolysis, cardiac arrhythmia, and lethal hyperthermia (26Nelson T.E. Curr. Mol. Med. 2002; 2: 347-369Crossref PubMed Scopus (80) Google Scholar, 27Treves S. Anderson A.A. Ducreux S. Divet A. Bleunven C. Grasso C. Paesante S. Zorzato F. Neuromuscul. Disord. 2005; 15: 577-587Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). This syndrome has been associated with a dysfunction of resting intracellular Ca2+ regulation. To date, about 112 mutations within the gene that codes for type 1 ryanodine receptor (RyR1) have been found on chromosome 19 (28Robinson R. Carpenter D. Shaw M.A. Halsall J. Hopkins P. Hum. Mutat. 2006; 27: 977-989Crossref PubMed Scopus (376) Google Scholar), and two mutations in the α1s subunit of the DHPR found on chromosome 1 have been associated with MH (29Jurkat-Rott K. McCarthy T. Lehmann-Horn F. Muscle Nerve. 2000; 23: 4-17Crossref PubMed Scopus (274) Google Scholar). To examine the possible role of abnormal sarcolemmal Ca2+ entry during MH, myotubes expressing mutations R615C, R2163C, and T48261, (collectively termed MHRyR1s) were exposed to supermaximal concentrations of KCl (60 mm K+) in the presence (Ca+) and absence (Ca–) of extracellular Ca2+. In addition, we also conducted experiments in the presence or absence of Ca2+ after the addition of NiCl2, nifedipine, La3+, or SKF 96365 in an attempt to identify, or if not to identify at least to characterize, the entry pathway. The results strongly suggest that the accentuated Ca2+ transients seen in myotubes expressing MHRyR1s after KCl depolarization that we previously reported (30Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) were the result of a more robust sarcolemmal Ca2+ entry and not an increased SR Ca2+ release in response to depolarization. This difference potentially plays a role in the pathophysiology of the MH syndrome. Construction and Expression of MHRyR1 cDNAs—A detailed explanation of construction and expression of the MHRyR1s (R615C, R2163C, and T48261) used in the present study has been previously described (30Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). All of the mutated and WTRyR1 cDNAs were packaged into HSV1 virions using a helper virus-free packaging system (31Fraefel C. Song S. Lim F. Lang P. Yu L. Wang Y. Wild P. Geller A.I. J. Virol. 1996; 70: 7190-7197Crossref PubMed Google Scholar, 32Wang Y. Fraefel C. Protasi F. Moore R.A. Fessenden J.D. Pessah I.N. DiFrancesco A. Breakefield X. Allen P.D. Am. J. Physiol. 2000; 278: C619-C626Crossref PubMed Google Scholar) and were then used to transduce dyspedic 1B5 myotubes (32Wang Y. Fraefel C. Protasi F. Moore R.A. Fessenden J.D. Pessah I.N. DiFrancesco A. Breakefield X. Allen P.D. Am. J. Physiol. 2000; 278: C619-C626Crossref PubMed Google Scholar) for 2 h at an multiplicity of infection of 0.5 and then cultured for 48 h prior to imaging. 1B5 cells (RyR-1, RyR-2, and RyR-3 null) were cultured on Matrigel-coated (BD Biosciences, San Jose, CA) 96-well plates (Opticlear® COSTAR 3614) as described previously (30Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Calcium Imaging of Myotubes—Differentiated myotubes were loaded with 5 μm Fluo-4AM (Molecular Probes Inc., Eugene, OR) at 37 °C, for 20 min in imaging buffer. The cells were then washed three times with imaging buffer (IB), transferred to a Nikon TE2000 microscope. Fluo-4 was excited at 494 nm with an argon light source, and fluorescence emission was measured at 516 nm using a 40 × 1.3na objective. The data were collected with an intensified 12-bit digital intensified CCD at 30 fps (Stanford Photonics, Stanford, CA) from regions consisting of 2–12 individual cells and analyzed using QED software (QED, Pittsburgh PA). We used the average fluorescence of the calcium transient (area under the curve) to compare responses. Individual response amplitudes were calculated in the following way: the cumulative fluorescence during the 30 s challenge (Ar) minus the average base-line fluorescence for the 10 s immediately prior to the challenge (Ab) was divided by Ab and then multiplied by 100. To compare different experiments, individual response amplitudes were normalized to the peak amplitude of the transient obtained in the same cell. The peak amplitude of a transient refers to the highest fluorescence value during the stimulation time minus the average base-line fluorescence for the 10 s immediately prior to the challenge. Solutions—Ionic compositions of Ca2+-replete imaging buffer (Ca+) with and without K+ (40 or 60 mm) are shown in Table 1. In KCl solutions, NaCl concentrations were adjusted to maintain a total ionic strength (Na+ concentration ([Na+]) + K+ concentration ([K+]) at 130 mm, but the product of [K+] × [Cl–] changes with different concentrations of KCl. In K2SO4 solutions, K2SO4 was used to increase [K+], and Na2SO4 was used to replace NaCl to maintain the [K+] × [Cl–] product at 670 and [Na+] + [K+] at 130 (the same values as Ca+). All nominally Ca2+-free buffers (Ca–) were prepared using the same protocol as the corresponding regular solutions but without CaCl2 added. Caffeine, NiCl2, nifedipine, LaCl3, and SKF96365 solutions were prepared by adding these compounds directly into either of the external buffers as well as the corresponding KCl solutions. Because of frequent trace Ca2+ contamination in other chemicals, residual free Ca2+ concentrations in Ca–(same composition as Ca+, but no Ca2+ added) and 60 mm Ca2+-free KCl solution (60KCl–Ca) were measured using Ca2+-selective microelectrodes and were ∼0.8 and ∼1.0 μm, respectively.TABLE 1Composition of buffer solutionsCa2+ replete buffer (Ca+)40 mm KCl (40KCl)60 mm KCl (60KCl)60 mm Ca2+-free KCl (60KCl-Ca)20 mm K2SO4 (20K2SO4)30 mm K2SO4 (30K2SO4)NaCl (mm)12590707012.757.17KCl (mm)5406060MgSO4 (mm)1.21.21.21.21.21.2Hepes (pH 7.4) (mm)252525252525Glucose (mm)666666CaCl2 (mm)22222Na2SO4 (mm)38.62531.415K2SO4 (mm)2030[Na+] + [K+]130130130130130130[K+] × [Cl-]670536080408040670670 Open table in a new tab Determination of Threshold for Maximum Ca2+ Transient in Response to KCl—From our previous studies we had determined that 50 mm [K+]o was sufficient to produce the maximum peak Ca2+ transient in myotubes (30Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). However, to make certain that our previous observations did not diverge from the measurements performed in the present study, we measured the magnitude of Ca2+ transients in myotubes expressing WT RyR1 produced by challenges with 40, 60, 80, and 100 mm [K+]o applied in a random order. Statistics—All of the values are expressed as the means ± S.E. Prism software (version 4.0b) was used for statistical analysis (GraphPad Software, San Diego, CA; www.graphpad.com). Student's t tests, one-way analysis of variance, and Tukey's multiple comparison tests were used to compare the response sizes and peak amplitudes. Effect of Extracellular Ca2+ on KCl-induced Ca2+ Transients—A significant observation in our previous study (30Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) was that dyspedic 1B5 myotubes expressing MHRyR1s consistently exhibited Ca2+ transients (area under the curve) in response to 60 mm KCl (60KCl) that were greater than those in myotubes expressing WTRyR1. The selection of 60 mm [K+]o to induce Ca2+ transients in WTRyR1 and MHRyR1 myotubes for this study was based both on our results obtained from the dose-response relationship measured in WT myotubes (Fig. 1) and previously published data (33Hodgkin A.L. Horowicz P. J. Physiol. 1960; 153: 386-403Crossref PubMed Scopus (253) Google Scholar, 34Caputo C. Bolanos P. J. Physiol. 1994; 481: 119-128Crossref PubMed Scopus (22) Google Scholar). The collective results from these studies show that in muscle fibers the amount of membrane depolarization caused by >50 mm [K+]o will elicit an elevation of myoplasmic [Ca2+] above the level required to saturate the contractile apparatus. Closer examination of data from our previous study revealed differences in the shapes of the Ca2+ transients induced by K+ between WTRyR1 and each of the seven MHRyR1s. Although there was no difference in the peak amplitude between the two groups, all MHRyR1 Ca2+ transients had a slower rate of decay than WTRyR1 Ca2+ transients (Fig. 1A, inset) and accounted for the greater areas under the Ca2+ transients elicited by 60KCl in myotubes expressing MHRyR1s. To determine whether Ca2+ entry contributed to the slower decline of the Ca2+ transient in myotubes expressing MHRyR1s during KCl depolarization, we increased the duration of depolarization from 10 s as it was in the previous study (30Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) to 30 s to record their rates of decay of the sustained phase of the transient and increased the interval between two 60KCl challenges from 50 s (30Yang T. Ta T.A. Pessah I.N. Allen P.D. J. Biol. Chem. 2003; 278: 25722-25730Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) to 3 min to permit the myotubes to return to their original resting intracellular free Ca2+ concentration between responses. As shown in Fig. 2A, the 1B5 myotubes expressing WTRyR1 or each of three common MHRyR1s (R615C, R2163C, and T4826I from RyR1 gene “hot spots” I, II, and III, respectively) were sequentially exposed to 60KCl, 60KCl–Ca, and 60KCl using the protocol described above. Consistent with our previous observation (2Protasi F. Franzini-Armstrong C. Allen P.D. J. Cell Biol. 1998; 140: 831-842Crossref PubMed Scopus (114) Google Scholar), all three types of MH myotubes demonstrated transients with much slower decay rates of the sustained phase of the transient during 60KCl depolarization compared with those expressing WTRyR1. Prior to exposure to 60KCl, the Ca+ replete buffer was exchanged with nominally Ca2+-free (Ca–) buffer for 5 s. The decay rate of the sustained phase of the Ca2+ transient during exposure to 60KCl–Ca was accelerated for all four types (one WT and three MH) of myotubes and was more rapid in all groups than the rate obtained from WTRyR1 depolarized in Ca+ buffer. On the other hand, the peak amplitude of the response was not significantly different for all three KCl exposures. In addition the ratio of the peak amplitude to the amplitude at 30 s in Ca2+-free medium (pure RyR1 Ca2+ release) was not different between cells expressing WTRyR1 compared with those expressing any of the three MHRyR1s. After 3 min of rest, the rate of decay of the sustained phase of the Ca2+ transient after the third 60KCl exposure was essentially the same as the first 60KCl response in both WTRyR1 and MHRyR1 myotubes. Therefore, the increased rate of decay seen during the 60KCl–Ca response was not the result of progressive store depletion in either group. Superimposing the first 60KCl response with the 60KCl–Ca response in the same myotube clearly showed the pronounced difference in their decay rate in the presence and the absence of external Ca2+. Importantly the relative increase in the decay rate in Ca–was much greater for all MHRyR1s than for WTRyR1. Comparison of the response sizes (Fig. 2B) demonstrated that 1) all three groups of MH myotubes had significantly larger total Ca2+ responses (p < 0.01) to 60KCl compared with WTRyR1, 2) the response sizes to 60KCl–Ca were significantly smaller than those to 60KCl in both WT and MH myotubes, and 3) the peak and total response sizes to 60KCl–Ca for WTRyR1 and MHRyR1 myotubes was the same. The ratio of the 60KCl–Ca response size to the 60KCl (Fig. 2C) response size for each of the three MHRyR1s was significantly (p < 0.01) less than that for WTRyR1 (42.6 ± 3.9, 48.6 ± 1.6, and 52.0 ± 3.3% for R615CRyR1, R2163CRyR1, and T4826IRyR1, respectively, versus 80.5 ± 4.6%, for WTRyR1), reflecting the difference in the rate of decay during depolarization for MHRyR1s (for superimposed normalized responses, see Fig. 2A). Because of the similarity in the ratios of these three MH mutations in their slower rate of decay compared with WTRyR1, we randomly picked T4826I for further investigation for the possible mechanisms for this difference. It is well known that extracellular [Cl–] also contributes to the membrane potential of frog muscle fibers when extracellular [K+] and [Cl–] were changed reciprocally to keep a constant product (35Hodgkin A.L. Horowicz P. J. Physiol. 1959; 148: 127-160Crossref PubMed Scopus (752) Google Scholar). In our 60 mm KCl solution both [K+] and [Cl–] were increased, substantially increasing the product of [K+] × [Cl–], compared with Ca+ buffers without KCl (Table 1). Thus it was theoretically possible that the responses to 60KCl we observed could have been affected by a Cl– overload. To rule this in or out, we exposed WTRyR1 and T4826IRyR1 myotubes to 60 mm KCl, whose [K+] × [Cl–] product in the buffer was the same as Ca+ buffer. Comparison of the shape and response sizes after depolarization with these two K+ solutions ([K+] × [Cl–] not constant versus [K+] × [Cl–] constant) showed that they were not significantly different from one another (Fig. 3A). It has also been demonstrated in single frog muscle fibers that removing extracellular Ca2+ to levels identical with that used for our Ca–experiments resulted in a slight membrane depolarization (9 ± 2 mV) and potentiation of twitch responses (12Caputo C. Gimenez M. J. Gen. Physiol. 1967; 50: 2177-2195Crossref PubMed Scopus (45) Google Scholar). Substitution of Ni2+ for missing Ca2+ in extracellular solutions resolved this problem (36Fischman D.A. Swan R.C. J. Gen. Physiol. 1967; 50: 1709-1728Crossref PubMed Scopus (14) Google Scholar). To examine this as a possible mechanism for the differences we observed between WTRyR1 and MHRyR1, we prepared 60KClNi solution by adding 2 mm NiCl2 to the 60KCl–Ca solution. Myotubes expressing T4826IRyR1 were exposed to 60KCl, 60KCl–Ca, 60KClNi and 60KCl sequentially (Fig. 3B). As observed before, the rate of decay of the Ca2+ transient in response to 60KCl–Ca depolarization was accelerated compared with the 60KCl response. Addition of 2 mm NiCl2 (60KCl–Ca+Ni) did not slow the accelerated rate of decay. However, the slow rate of decay of the sustained phase of KCl responses in T4826IRyR1 myotubes was restored in Ca+ buffer (Fig. 3B). Effects of Ca2+ Entry Blockers on Depolarization-induced Ca2+ Entry—To better define the mechanism for the enhanced Ca2+ entry caused by depolarization of cells with MHRyR1 mutations, we used a pharmacologic library to attempt to block the response. To do this, we chose (0.1 mm) lanthanum chloride (La3+; a nonspecific Ca2+ entry blocker (37Goodman F.R. Weiss G.B. Arch. Int. Pharmacodyn. Ther. 1974; 209: 14-25PubMed Google Scholar)), 20 μm SKF 96365 (a SOCE and TRP channel blocker (38Leung Y.M. Kwan C.Y. Jpn. J. Pharmacol. 1999; 81: 253-258Crossref PubMed Scopus (57) Google Scholar) that has also been shown to block cation influx attributable to ECCE (24Cherednichenko G. Hurne A.M. Fessenden J.D. Lee E.H. Allen P.D. Beam K.G. Pessah I.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15793-15798Crossref PubMed Scopus (99) Google Scholar)), and 1 μm nifedipine (the selective L-type Ca2+ channel blocker (39Chen W. Hui C.S. J. Physiol. 1991; 444: 579-603Crossref PubMed Scopus (20) Google Scholar). As shown in Fig. 4A, La3+ (0.1 mm) added 10 s after 60KCl exposure immediately accelerated the decay rate of the sustained phase of the Ca2+ transients for both WTRyR1 and T4826IRyR1 myotubes (responses labeled #2 for WTRyR1 and T4826IRyR1). The 60KCl responses (responses labeled #3) with La3+ (0.1 mm) added 5 s before KCl exposure demonstrated a more significantly accelerated decay rate of the sustained phase for both WT and T4826IRyR1 myotubes, which is better illustrated by superimposed normalized traces on the right side of Fig. 4A. The last 60KCl responses (responses labeled #4) in the absence of extracellular La3+ partially restored the slower decay rate of the sustained phase in both WTRyR1 and T4826IRyR1 myotubes, although the recovery was not complete compared with the #1 responses. The ratio of response sizes of 60KCl + La3+ (responses labeled #3) to 60KCl (responses labeled #1) (Fig. 4B) is significantly smaller (p < 0.01) for T4826I (64.7 ± 3.1%) compared with WT (85.3 ± 3.6%), indicating that a significantly larger portion of the 60KCl response in T4826IRyR1 myotubes was diminished by La3+ compared with WTRyR1. Of note, no change in the peak amplitude of the KCl responses was observed with the sequential KCl stimulations. The decay rate of the sustained phase of the Ca2+ transient during KCl depolarization was drastically accelerated in both WT and T4826I myotubes by 20 μm SKF 96365 (responses labeled #2 in Fig. 5A). Removing SKF 96365 prior to the next 60KCl stimulation restored the slower decay of sustained phase to pre-exposure rates in T4826I myotubes. 20 μm of SKF 96365 appears to accelerate the decay rate instantaneously (Fig. 5A, inset), and the ratios of response #2 to response #1 sizes for WT and T4826I are 72.8 ± 3.2 and 49.0 ± 3.9%, respectively, which suggests a more significant effect (p < 0.01) of SKF 96365 on T4826I myotubes compared with WT myotubes (Fig. 5). Interestingly, the response #2 to response #1 ratios for the SKF 96365 effect tended to be smaller than even those for 60KCl–Ca shown in Fig. 2 (80.5 ± 4.6 and 52.0 ± 3.3% for WT and T4826I, respectively). Nifedipine administered 5 s before 60KClNif (60KCl containing 1 μm nifedipine) stimulation (#2 responses in Fig. 6A) had no significant effect on the sustained phase in either WT or MH myotubes compared with the corresponding control #1 responses. Caffeine-induced Ca2+ Transient Is Not Mediated by Sarcolemmal Ca2+ Entry—To investigate whether the presence of extracellular Ca2+ has any effect on the Ca2+ transient elicited by direct RyR1 activators, 20 mm caffeine was used to stimulate Ca2+ release from SR from myotubes expressing WTRyR1 and T4826IRyR1, using the same experimental protocol (Fig. 7A). In contrast to the KCl responses shown before, the responses to regular 20 mm caffeine (20caff) and Ca2+-free 20 mm caffeine (20caff–Ca) were identical in both WTRyR1 and T4826IRyR1 myotubes. These similarities were clearly illustrated when the normalized data were superimposed. Interestingly, the peak amplitude of the caffeine responses decreased significantly with sequential caffeine successive caffeine challenges and was independent of external Ca2+ (Fig. 7B). As a result, the response sizes showed a similar decrease in both WTRyR1 and T4826IRyR1 myotubes (Fig. 7C). When the peak amplitudes and response sizes for the first 20caff response and the 20caff–Ca response were superimposed (Fig. 7D), the decreases in peak amplitude were coincident with the decrease in response size (area under the curve) for both genotypes, suggesting that the decreased response size to 20caff–Ca resulted from decreased peak amplitude rather than a change in the sustained phase. These observations are also born out when the cells are stimulated with caffeine in the presence of SKF 96365. (Fig. 7E). Neither W" @default.
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• W1967440360 title "Enhanced Excitation-coupled Calcium Entry in Myotubes Is Associated with Expression of RyR1 Malignant Hyperthermia Mutations" @default.
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