Correspondence to: Robert T. Dirksen, Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642. Fax:716-273-2652 E-mail:robert_dirksen{at}urmc.rochester.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
L-type Ca2+ channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H.T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:7275.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and "RyR1-knockout" (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Qmax, 7.5 ± 0.8 and 4.5 ± 0.4 nC/µF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (Gmax/Qmax). Thus, RyR1 enhances both the expression level and Ca2+ conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (Afast and Aslow) and time constants (slow and
fast) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; Afast/[Afast + Aslow] ~ 0.25). However, both
fast and
slow were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (
fast, 8.5 ± 1.2 and 4.4 ± 0.5 ms;
slow, 79.5 ± 10.5 and 34.6 ± 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (-) Bay K 8644 (5 µM) caused a hyperpolarizing shift (~10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both Aslow and Afast in normal myotubes, but a large increase in only Afast in dyspedic myotubes. Equimolar substitution of Ba2+ for extracellular Ca2+ increased both Afast and Aslow in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either Afast or Aslow. These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca2+ channel accessory subunit.
Key Words: excitationcontraction coupling, skeletal muscle, charge movement
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Skeletal and cardiac muscle dihydropyridine receptors (DHPRs)1 are voltage-dependent L-type calcium channels (L-channels) (
Interestingly, the presence of the RyR1 protein promotes the Ca2+ conducting activity and accelerates the activation of skeletal L-channels ("retrograde signal") (
Interaction between intracellular signaling molecules, such as G-proteins (for review, see
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of Normal and Dyspedic Myotubes
Primary cultures of myotubes were prepared from skeletal muscle of newborn normal and dyspedic mice as described previously (
|
Electrophysiologic Measurements
Whole-cell patch-clamp experiments were carried out at room temperature (2022°C), 711 d after the initial plating of myoblasts. For all experiments, the holding potential was -80 mV. T-type Ca2+ currents were eliminated by a conditioning prepulse consisting of a 1-s depolarization to -20 mV followed by a 25-ms repolarization to -50 mV before each test pulse ( (n = 82), and the voltage error due to series resistance (Ve = Rs x ICa) was less than ~5 mV (for these experiments, the average was 2.66 ± 0.19 mV, n = 82). The average time constant for charging the membrane capacitance (
m = Rs x Cm) was 0.37 ± 0.02 ms (n = 82) and was never larger than 1.21 ms. All data are presented as mean ± SEM.
Macroscopic Calcium Currents
The whole-cell variant of the patch clamp technique ( when filled with internal solution (see below). Peak inward Ca2+ currents were assessed at the end of 200-ms test pulses of variable amplitude and plotted as a function of the membrane potential (I-V curves). I-V curves were subsequently fitted according to:
![]() |
(1) |
where Vrev is the extrapolated reversal potential of the calcium or barium current, V is the membrane potential during the test pulse, I is the peak current during the test pulse, Gmax is the maximum L-channels conductance, VG1/2 is the voltage for half activation of Gmax, and kG is the slope factor. The activation phase of macroscopic ionic currents was fitted using one of the following exponential functions (Equation 2 and Equation 3):
![]() |
(2) |
![]() |
(3) |
where I(t) is the current at time t after the depolarization, A0, Afast, and Aslow are the steady state current amplitudes of each component with their respective time constants of activation (0,
fast, and
slow), and C represents the steady state peak current. In all cases, the fitting procedure started at the zero current level, which corresponded to 57 ms after the initiation of the voltage pulse (>10 x
m). This approach limited artifacts introduced by the declining phase of Qon, since the magnitude of Qon reaches >90% of its maximal value before this time (see Fig 1). In addition, L-currents were also recorded before and after ionic current blockade with 0.5 mM Cd2+ + 0.2 mM La3+ in a separate set of experiments. The Cd2+/La3+-sensitive currents lacked intramembrane charge movements and exhibited nearly identical activation kinetics as those obtained before gating current subtraction (data not shown). Thus, the fast component of L-current activation described in this study is not greatly influenced by the declining phase of the Qon gating current transient.
|
Intramembrane Charge Movement
Immobilization-resistant intramembrane charge movements were measured in whole-cell mode by a method described previously (
![]() |
(4) |
where VQ1/2 and kQ have their usual meanings with regard to charge movement.
Recording Solutions
For measurements of macroscopic ionic and gating currents, the internal solution consisted of (mM): 140 Cs-aspartate, 10 Cs2-EGTA, 5 MgCl2, and 10 HEPES, pH 7.40 with CsOH. Macroscopic calcium currents were recorded in an external solution containing (mM): 145 TEA-Cl, 10 CaCl2, 0.003 TTX (Alomone Laboratories), and 10 HEPES, pH 7.40 with TEA-OH. For measurements of macroscopic barium currents, 10 mM BaCl2 was substituted for the 10 mM CaCl2 in the external solution. The external calcium current recording solution was supplemented with 0.5 mM CdCl2 + 0.2 mM LaCl3 for measurements of intramembrane charge movement. For some experiments, the influence of RyR1 on the stimulatory action of (-) Bay K 8644 (0.005 mM), a pure DHP agonist, was evaluated after addition to the external calcium current recording solution. Except where noted, all chemical reagents were obtained from Sigma Chemical Co.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dyspedic Myotubes Exhibit a Reduction in Immobilization-resistant Charge Movement
If the gating charge moved is similar for channels with different Pos, then the density of channel proteins can be estimated from the magnitude of the maximal immobilization-resistant intramembrane charge movement (Qmax) (
Null mutations of a single allele in a diploid organism could potentially result in a reduced probability of gene expression, and thus lead to haploinsufficiency. Therefore, we have investigated whether the presence of a single wild-type RyR1 allele is sufficient to supply the number of ryanodine receptors required to sustain normal L-channel activity. With this in mind, normal myotubes were genotyped and divided into two groups: homozygous normal (+/+) and heterozygous normal (+/-) for the wild-type RyR1 allele. However, no significant (P > 0.4) differences were found in the voltage-dependent parameters of either L-channel conductance or charge movement (Table 1) between these two genotypic groups. These data are in agreement with morphological studies that indicate that muscle fibers obtained from homozygous and heterozygous normal embryos are structurally indistinguishable (
Dyspedic myotubes exhibit a very modest amount of slowly activating L-current, which is greatly enhanced 24 d after nuclear injection of wild-type RyR1 cDNA (
|
RyR1 Alters Two Kinetic Components of L-Current Activation
The slow activation time course of skeletal L-current in cultured mouse myotubes has often been approximated by fitting the data to a single exponential function (2 < 0.005) quantitative description of activation in BC3H1 cells requires two exponential components (
2) the description of L-current activation (Fig 3 A, right). The biexponential nature of the activation of the calcium current is best appreciated by replotting the normalized currents on a semilogarithmic scale (Fig 3 A, bottom). The use of three exponential components did not significantly improve the fit of the data. The activation time course of the residual calcium current recorded from dyspedic myotubes was also best described by the sum of two exponential components (data not shown).
|
The second order exponential fitting procedure resulted in the extraction of the steady state amplitudes (Afast and Aslow) and corresponding activation time constants (fast and
slow) that comprise the macroscopic L-current. The influence of RyR1 on the amplitudes and time constants of macroscopic L-currents at +40 mV is summarized in Fig 3BD. Amplitudes and time constants were compared between normal (N), dyspedic (Y), and RyR1-expressing (R) dyspedic myotubes after 200-ms depolarizations to +40 mV, a potential at which L-current conductance is maximal (
fast and
slow (Fig 3 D). Nuclear injection of dyspedic myotubes with wild-type RyR1 cDNA resulted in a marked increase in both Afast and Aslow and also a corresponding increase in
fast and
slow (Fig 3, BD). Thus, the parallel dependence of both the amplitudes and time constants of the macroscopic L-current on the presence of RyR1 indicates that skeletal L-channels in cultured myotubes exhibit two distinct kinetic gating modes, each of which are modified by interaction with RyR1.
Bay K 8644 Accelerates Dyspedic L-Current Activation through a Preferential Increase in Afast
The data illustrated in Table 1 and Fig 3 suggest that RyR1 influences the functional conformation of the skeletal L-channel. Since DHP modulation of the activity and voltage dependence L-channels is state dependent (
|
|
As demonstrated in Fig 3 A, L-current activation was clearly best described by the sum of two exponential functions, and this becomes even more evident in the presence of DHP (where the second-order fit reduced 2 >43-fold, Fig 4 A, left). This analysis revealed that at +40 mV the DHP-induced acceleration in L-channel activation kinetics in normal myotubes arises primarily from a preferential increase in the amplitude of the fast component (Afast) of L-current (Fig 4 B, left). Specifically, (-) Bay K 8644 caused a significant increase (83 ± 12%, n = 6) in the magnitude of Afast, without significantly altering Aslow. However, the time constants of each component (
fast and
slow) were not significantly altered in the presence of the DHP agonist (Fig 4 B, right). In dyspedic myotubes at +40 mV, (-) Bay K 8644 induced an increase in Afast (174 ± 30%, n = 7) without significantly altering the magnitudes of Aslow,
fast, or
slow (Fig 4 C). Consequently, in dyspedic myotubes, the DHP agonist increased the relative contribution of Afast to the total L-current (at +40 mV, Afast/[Afast + Aslow] was 0.24 ± 0.02 in control and 0.49 ± 0.02 in 5 µM (-) Bay K 8644, P < 0.001). Thus, at +40 mV, (-) Bay K 8644 preferentially augmented the magnitude of Afast in both normal and dyspedic myotubes without altering Aslow,
fast, or
slow.
The data described in Fig 4 were obtained for test pulses to +40 mV, a potential at which L-channel conductance is maximal in both the presence and absence of DHP agonist. The effects of (-) Bay K 8644 (5 µM) on the voltage dependence of macroscopic skeletal L-currents and its component parts (Afast and Aslow) are summarized in Fig 5. The peak I-V relationships of normal (Fig 5 A) and dyspedic (C) myotubes were obtained in the absence () and presence () of (-) Bay K 8644 (5 µM). In both types of myotubes, the DHP agonist produced both a similar increase in total L-current density and a hyperpolarizing shift (~10 mV, Table 2) in the macroscopic I-V relation. However, these effects on the overall macroscopic currents arise from qualitatively distinct alterations in the two kinetic components of skeletal L-current. In Fig 5, the magnitudes of Afast and Aslow in normal (B) and dyspedic (D) myotubes were normalized by the average peak control value for each data set (Aslow and Afast were: 11.89 ± 1.10 and 2.30 ± 0.40 pA/pF for normal myotubes, and 1.23 ± 0.16 and 0.47 ± 0.06 pA/pF for dyspedic myotubes). This analysis revealed that, even under control conditions, Afast activates ~10 mV more hyperpolarized than Aslow. In normal myotubes, (-) Bay K 8644 increased (up to approximately twofold) the normalized magnitude of both Afast and Aslow. In addition, the DHP-induced hyperpolarizing shift in the voltage dependence of L-channel activation is reflected as a selective shift in the voltage dependence of Aslow. Interestingly, (-) Bay K 8644 produced an approximately threefold increase in Afast in the absence of an alteration in the magnitude of Aslow in dyspedic myotubes. The remarkable DHP insensitivity of Aslow in dyspedic myotubes indicates that the RyR1 protein imparts a strong influence on the state-dependent action of DHP agonists on skeletal L-channels.
|
RyR1 Modifies Skeletal L-Channel Divalent Conductance
Equimolar substitution of Ba2+ for extracellular Ca2+ produces an increase in peak L-current and a hyperpolarizing shift in the voltage dependence of channel activation in cardiac muscle (
|
The relative contribution of Afast and Aslow to the macroscopic Ca2+ and Ba2+ currents (Fig 6 B, b and d) were extracted by fitting a second-order exponential function to the activation phase of the ionic currents (Fig 6 A). Substitution of Ba2+ for extracellular Ca2+ increased both Afast and Aslow in normal myotubes (Fig 6 B, b) without greatly altering either fast or
slow (data not shown). The identical divalent substitution failed to significantly alter the magnitude of either Afast or Aslow in dyspedic myotubes (Fig 6 B, d). Nevertheless, extracellular Ba2+ substitution caused a hyperpolarizing shift in the voltage dependence of both Afast and Aslow in normal and dyspedic myotubes. Thus, the skeletal muscle ryanodine receptor appears to influence relative L-channel divalent conductance, but not the differential ability of Ca2+ and Ba2+ ions to modify external surface charge. These data suggest that the interaction of RyR1 with the skeletal muscle DHPR may exert long-range effects on the functional conformation of the pore of the skeletal L-channel. However, a direct evaluation of the effects of RyR1 on skeletal L-channel selectivity must await a systematic determination of the monovalent and divalent permeability sequence of L-channels in normal and dyspedic myotubes.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In skeletal muscle, the coupling of sarcolemmal depolarization to the release of SR calcium is thought to involve a direct physical interaction between the DHPR and the RyR1. If the interaction of RyR1 with the DHPR stabilizes certain L-channel conformational states by altering transition rates between states, then disruption of this interaction may result in altered L-channel function. However, no study has systematically characterized the influence of RyR1 on the biophysical and pharmacological properties of the skeletal L-current. Consequently, we have evaluated the influence of RyR1 on the expression level, voltage dependence, activation rate, DHP modulation, and divalent conductance of the skeletal L-channel. Our results indicate that the density of functional sarcolemmal L-channels is reduced by ~40% in dyspedic myotubes compared with that of myotubes derived from their phenotypically normal littermates. We also demonstrated that dyspedic L-currents exhibit accelerated activation (fast and
slow), a greater separation between Q-V and G-V relationships ([VG1/2 - VQ1/2] was 16.7, 33.7, and 13.5 mV for normal, dyspedic, and RyR1-expressing dyspedic myotubes, respectively), and similar macroscopic conductances to Ca2+ and Ba2+. The ability of RyR1 to enhance the coupling between charge movement and pore opening (i.e., reduce Q-V/G-V separation) and alter the relative conductance to divalent ions (Ca2+ versus Ba2+) suggests that RyR1 imparts long-range effects on the conformational state of the pore of the skeletal L-channel. In addition, (-) Bay K 8644 increased the magnitude of both the fast (Afast) and slow (Aslow) components of the total L-current in normal myotubes, but only enhanced the fast component in dyspedic myotubes. Thus, our results indicate that the presence of RyR1 in skeletal muscle imparts a strong influence on several essential properties of the skeletal L-channel.
RyR1 Promotes L-Channel Expression
Previous studies have demonstrated that dyspedic muscle exhibits a 2550% reduction in total DHP binding capacity compared with normal muscle (
Two Kinetic Components of L-Current Activation
The activation kinetics of skeletal L-currents in cultured myotubes has often been approximated by fitting the activation time course to a single exponential function. This approach has provided a convenient means for making qualitative comparisons in channel kinetics under different conditions (1 = 220 ms and
2 = 10200 ms) similar to those reported here. Moreover, the voltage dependence and relative contribution of the faster component in BC3H1 cells (~25% at +40 mV) is comparable with our results using primary myotube cultures. Our analyses indicate that while a single exponential often results in a reasonable approximation of L-channel activation at strong depolarizations (>20 mV), two activation terms are clearly required under conditions that enhance the relative contribution of the fast component of L-current activation. An increase in the relative contribution of Afast results in a clear bi-exponential activation for L-currents activated at threshold potentials (e.g., -10 mV) and for L-currents in dyspedic myotubes treated with (-) Bay K 8644 (Fig 4 A). In addition, we have recently reported that prolonged depolarization markedly increases the relative contribution of Afast to the total L-current, resulting in a clearly bi-exponential L-channel activation time course (
Apparent channel activation can be altered when a significant degree of inactivation occurs during the activation process. Thus, inactivation occurring during our 200-ms test pulses could influence the kinetic properties of L-channel activation (Afast, fast, Aslow, and
slow) reported here. Since we have not systematically characterized inactivation in normal and dyspedic myotubes in this study, we cannot rule out a possible contribution of inactivation to the effects of RyR1 on the kinetic properties of L-channel activation. However, any effects of inactivation on apparent L-channel activation would be anticipated to be minimal in our experiments since inactivation of L-currents is very slow (~25x slower than
slow described here) in myotubes (
Normal myotubes possess T-type Ca2+ channels (
Dysgenic myotubes, which lack an intact gene for the skeletal muscle DHPR, exhibit a rapidly activating, DHP-sensitive L-current (Idys; 1C subunit. It is also likely that the channels that account for Idys make a significant contribution to the residual immobilization-resistant intramembrane charge movement found in dysgenic myotubes (Qdys;
act ~ 5 ms), and are augmented two- to threefold by (-) Bay K 8466. However, several findings suggest that the fast component of skeletal L-current activation is distinct from Idys. For example, Idys is more strongly stimulated by (-) Bay K 8644 than L-current in normal myotubes (
It is uncertain whether the two components of L-channel activation in mouse skeletal myotubes reflect the gating of separate ion channels or two gating modes of a single Ca2+ channel protein. However, our data support the latter possibility since RyR1 regulates several properties of these two components in a quantitatively similar manner. For example, Afast and Aslow are each reduced approximately sevenfold in the absence of RyR1, resulting in a constant relative contribution of fast and slow components to the total L-current in both normal and dyspedic myotubes. Interestingly, RyR1 expression increased Afast to a value comparable with that of normal myotubes and only partially restored Aslow, resulting in a moderately significant (P < 0.05) increase in the relative contribution of Afast. In addition, both fast and
slow are approximately twofold faster in dyspedic myotubes, and expression of RyR1 in dyspedic myotubes restores both
fast and
slow to values similar to those of normal myotubes. Based on single channel recordings (
" in the model of
Distribution and Targeting of L-Channels in Dyspedic and RyR1-expressing Dyspedic Myotubes
Skeletal muscle dihydropyridine receptors and ryanodine receptors colocalize in clusters that are randomly distributed in punctate foci throughout the muscle cell (
Our results, in which injection with RyR1 cDNA restores both the ICa/Qmax (Fig 2) and Gmax/Qmax (Table 1) ratios of dyspedic myotubes to values comparable with those of normal myotubes, indicate that the vast majority of dyspedic sarcolemmal L-channels are functionally "recoupled" upon RyR1 expression. Thus, it is possible that the expressed RyR1 proteins are efficiently targeted to the majority of junctions throughout the injected dyspedic myotubes. However,
RyR1 Acts as an Allosteric Modulator of Skeletal L-Channel Activity
The functional properties of voltage-dependent Ca2+ channels are markedly influenced by direct interactions with auxiliary Ca2+ channel subunits and intracellular signaling proteins. With regard to L-channels, ß-subunits augment peak Ca2+ current by increasing the number of channels in the surface membrane (1-subunits to the plasma membrane (
2-
-subunit generally acts to potentiate the effects of ß subunits on L-current amplitude and kinetics (
2-
- and ß-subunits enhance DHP binding to L-channels (
Several important intracellular signaling molecules are also known to interact and modify the functional properties of voltage-dependent Ca2+ channels. For example, the properties of neuronal N- and P/Q-type Ca2+ channels are modulated by interaction with synaptic membrane proteins (e.g., syntaxin and the 25-kD synaptosome-associated protein, SNAP25) that control vesicle docking and membrane fusion during neurotransmitter release (for review, see -subunits directly inhibit neuronal N-, P/Q-, and R-type Ca2+ channels by reducing current amplitude, slowing channel activation, and shifting channel activation to more depolarized potentials (for review, see
![]() |
Footnotes |
---|
Portions of this work were previously published in abstract form (Avila, G., and R.T. Dirksen. 2000. Biophys. J. 78:427a).
1 Abbreviations used in this paper: DHPR, dihydropyridine receptor; EC, excitationcontraction; L-channel, L-type Ca2+ channel; RyR, ryanodine receptor; SR, sarcoplasmic reticulum.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Drs. Kurt G. Beam and Paul D. Allen for providing us access to the dyspedic mice used in this study, as well as for their advice and continued support. We also thank Dr. Ted Begenisich for helpful discussions and comments on the manuscript and Linda Groom for excellent technical assistance.
This work was supported by National Institutes of Health grant AR44657 (R.T. Dirksen), a Neuromuscular Disease Research grant (R.T. Dirksen), and CONACYT postdoctoral fellowship 990236 (G. Avila).
Submitted: 7 December 1999
Revised: 22 February 2000
Accepted: 23 February 2000
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, B.A., Tanabe, T., Mikami, A., Numa, S., Beam, K.G. 1990. Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs. Nature. 346:569-572[Medline].
Armstrong, C.M., Bezanilla, F.M., Horowicz, P. 1972. Twitches in the presence of ethylene glycol bis(-aminoethyl ether)-N,N'-tetraacetic acid. Biochim. Biophys. Acta. 267:605-608[Medline].
Beurg, M., Sukhareva, M., Ahren, C.A., Conklin, M.W., Perez-Reyes, E., Powers, P.A., Gregg, R.G., Coronado, R. 1999. Differential regulation of skeletal muscle L-type Ca2+ current and excitationcontraction coupling by the dihydropyridine receptor ß1 subunit. Biophys. J 76:1744-1756
Beurg, M., Sukhareva, M., Strube, C., Powers, P.A., Gregg, R.G., Coronado, R. 1997. Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor ß1 subunit transfected with ß1 cDNA. Biophys. J 73:807-818[Abstract].
Block, B.A., Imagawa, T., Campbell, K.P., Franzini-Armstrong, C. 1988. Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle. J. Cell Biol. 107:2587-2600[Abstract].
Buck, E.D., Nguyen, H.T., Pessah, I.N., Allen, P.D. 1997. Dyspedic mouse skeletal muscle expresses major elements of the triadic junction, but lacks detectable ryanodine receptor protein and function. J. Biol. Chem. 272:7360-7367
Caffrey, J.M. 1994. Kinetic properties of skeletal-musclelike high-threshold calcium currents in a non-fusing muscle cell line. Pflügers Arch. 427:277-288.
Catterall, W.A. 1999. Interactions of presynaptic Ca2+ channels and SNARE proteins in neurotransmitter release. Ann. NY Acad. Sci. 868:144-159
Chavis, P., Fagni, L., Lansman, J.B., Bockaert, J. 1996. Functional coupling between ryanodine receptors and L-type calcium channels in neurons. Nature. 382:719-722[Medline].
Dirksen, R.T., Beam, K.G. 1995. Single calcium channel behavior in native skeletal muscle. J. Gen. Physiol. 105:227-247[Abstract].
Dirksen, R.T., Beam, K.G. 1996. Unitary behavior of skeletal, cardiac, and chimeric L-type Ca2+ channels expressed in dysgenic myotubes. J. Gen. Physiol. 107:731-742[Abstract].
Dirksen, R.T., Beam, K.G. 1999. Role of calcium permeation in dihydropyridine receptor function: insights into channel gating and excitationcontraction coupling. J. Gen. Physiol. 114:393-404
Dolphin, A.C. 1998. Mechanisms of modulation of voltage-dependent calcium channels by G-proteins. J. Physiol. 506:3-11
Fleig, A., Takeshima, H., Penner, R. 1996. Absence of Ca2+ current facilitation in skeletal muscle of transgenic mice lacking the type 1 ryanodine receptor. J. Physiol. 496:339-345[Abstract].
Flucher, B.E., Andrews, S.B., Fleischer, S., Marks, A.R., Caswell, A., Powell, J.A. 1993. Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro. J. Cell. Biol. 123:1161-1174[Abstract].
Fox, A.P., Nowycky, M.C., Tsien, R.W. 1987. Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones. J. Physiol. 394:149-172[Abstract].
Franzini-Armstrong, C., Jorgensen, A.O. 1994. Structure and development of E-C coupling units in skeletal muscle. Annu. Rev. Physiol. 56:509-534[Medline].
Franzini-Armstrong, C., Pincon-Raymond, M., Rieger, F. 1991. Muscle fibers from dysgenic mouse in vivo lack a surface component of peripheral couplings. Dev. Biol. 146:364-376[Medline].
Garcia, J., Beam, K.G. 1994. Measurement of calcium transients and slow calcium current in myotubes. J. Gen. Physiol. 103:107-123[Abstract].
Garcia, J., Tanabe, T., Beam, K.G. 1994. Relationship of calcium transients to calcium currents and charge movements in myotubes expressing skeletal and cardiac dihydropyridine receptors. J. Gen. Physiol. 103:125-147[Abstract].
Grabner, M., Dirksen, R.T., Suda, N., Beam, K.G. 1999. The II-III loop of the skeletal muscle dihydropyridine receptor is responsible for the bi-directional coupling with the ryanodine receptor. J. Biol. Chem. 274:21913-21919
Graef, I.A., Mermelstein, P.G., Stankunas, K., Neilson, J.R., Deisseroth, K., Tsien, R.W., Crabtree, G.R. 1999. L-type calcium channels and GSK-3 regulate the activity of NF-Atc4 in hippocampal neurons. Nature. 401:703-708[Medline].
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391:85-100[Medline].
Harasztosi, C., Sipos, I., Kovacs, L., Melzer, W. 1999. Kinetics of inactivation and restoration of the L-type calcium current in human myotubes. J. Physiol. 516:129-138
Hille, B. 1992. Ionic Channels of Excitable Membranes. 2nd ed Sunderland, MA, Sinauer Associates, Inc, pp. 607.
Kass, R.S., Sanguinetti, M.C. 1984. Inactivation of calcium channel current in the calf cardiac Purkinje fiber: evidence for voltage- and calcium-mediated mechanisms. J. Gen. Physiol. 84:705-726[Abstract].
Lacerda, A.E., Kim, H.S., Ruth, P., Perez-Reyes, E., Flockerzi, V., Hofmann, F., Birnbaumer, L., Brown, A.M. 1991. Normalization of current kinetics by interaction between the a1 and b subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel. Nature. 352:527-530[Medline].
Lorenzon, N.M., Grabner, M., Beam, K.G. 1999. N-terminal tagging of the skeletal ryanodine receptor with green fluorescent protein does not interfere with the restoration of bi-directional coupling. Biophys. J. 76:A304. (Abstr.).
McPherson, P.S., Campbell, K.P. 1993. The ryanodine receptor/Ca2+ release channel. J. Biol. Chem. 268:13765-13768
Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., Numa, S. 1989. Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature. 340:230-233[Medline].
Nabauer, M., Callewaert, G., Cleemann, L., Morad, M. 1989. Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes. Science. 244:800-803[Medline].
Nakai, J., Dirksen, R.T., Nguyen, H.T., Pessah, I.N., Beam, K.G., Allen, P.D. 1996. Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor. Nature. 380:72-75[Medline].
Nakai, J., Ogura, T., Protasi, F., Franzini-Armstrong, C., Allen, P.D., Beam, K.G. 1997. Functional nonequality of the cardiac and skeletal ryanodine receptors. Proc. Natl. Acad. Sci. USA. 94:1019-1022
Nakai, J., Sekiguchi, N., Rando, T.A., Allen, P.D., Beam, K.G. 1998b. Two regions of the ryanodine receptor involved in coupling with L-type Ca2+ channels. J. Biol. Chem. 273:13403-13406
Nakai, J., Tanabe, T., Konno, T., Adams, B., Beam, K.G. 1998a. Localization in the II-III loop of the dihydropyridine receptor of a sequence critical for excitationcontraction coupling. J. Biol. Chem. 273:24983-24986
Neely, A., Wei, X., Olcese, R., Birnbaumer, L., Stefani, E. 1993. Potentiation by the ß subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel. Science 262:575-578[Medline].
O'Connell, K.M.S., Dirksen, R.T. 2000. Prolonged depolarization accelerates activation of the L-type Ca2+ current (L-current) in mouse skeletal myotubes. Biophys. J. 78:370a. (Abstr.).
Peterson, B.Z., DeMaria, C.D., Yue, D.T. 1999. Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type calcium channels. Neuron 22:549-558[Medline].
Ríos, E., Brum, G. 1987. Involvement of dihydropyridine receptors in excitationcontraction coupling in skeletal muscle. Nature. 325:717-720[Medline].
Schneider, M.F., Chandler, W.K. 1973. Voltage-dependent charge movement of skeletal muscle: a possible step in excitationcontraction coupling. Nature. 242:244-246[Medline].
Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F., Dascal, N. 1991. The roles of the subunits in the function of the calcium channel. Science 253:1553-1557[Medline].
Shistik, E., Ivanina, T., Puri, T., Hosey, M., Dascal, N. 1995. Ca2+ current enhancement by 2/
and ß subunits in Xenopus oocytes: contributions of changes in channel gating and
1 protein level. J. Physiol. 489:55-62[Abstract].
Strube, C., Beurg, M., Powers, P.A., Gregg, R.G., Coronado, R. 1996. Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor ß1 subunit. Biophys. J. 71:2531-2543[Abstract].
Strube, C., Beurg, M., Sukhareva, M., Ahern, C.A., Powell, J.A., Powers, P.A., Gregg, R.G., Coronado, R. 1998. Molecular origin of the L-type Ca2+ current of skeletal muscle myotubes selectively deficient in dihydropyridine receptor ß1a subunit. Biophys. J. 75:207-217
Takekura, H., Franzini-Armstrong, C. 1999. Correct targeting of dihydropyridine receptors and triadin in dyspedic mouse skeletal muscle in vivo. Dev. Dyn. 214:372-380[Medline].
Takekura, H., Nishi, M., Noda, T., Takeshima, H., Franzini-Armstrong, C. 1995. Abnormal junctions between surface membrane and sarcoplasmic reticulum in skeletal muscle with a mutation targeted to the ryanodine receptor. Proc. Natl. Acad. Sci. USA. 92:3381-3385[Abstract].
Takeshima, H., Iino, M., Takekura, H., Nishi, M., Kuno, J., Minowa, O., Takano, H., Noda, T. 1994. Excitationcontraction uncoupling and muscular degeneration in mice lacking functional skeletal muscle ryanodine-receptor gene. Nature. 369:556-559[Medline].
Tanabe, T., Adams, B.A., Numa, S., Beam, K.G. 1991. Repeat I of the dihydropyridine receptor is critical in determining calcium channel activation kinetics. Nature. 352:800-803[Medline].
Tanabe, T., Beam, K.G., Adams, B.A., Niidome, T., Numa, S. 1990. Regions of the skeletal muscle dihydropyridine receptor critical for excitationcontraction coupling. Nature. 346:567-569[Medline].
Tanabe, T., Beam, K.G., Powell, J.A., Numa, S. 1988. Restoration of excitationcontraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature. 336:134-139[Medline].
Varadi, G., Lory, P., Schultz, D., Varadi, M., Schwartz, A. 1991. Acceleration of activation and inactivation by the b subunit of the skeletal muscle calcium channel. Nature 352:159-162[Medline].
Wei, X., Perez-Reyes, E., Lacerda, A.E., Schuster, G., Brown, A.M., Birnbaumer, L. 1991. Heterologous regulation of the cardiac calcium channel 1 subunit by skeletal muscle b and g subunits. J. Biol. Chem. 266:21943-21947