Correspondence to: Kurt G. Beam, Department of Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523. Fax:Fax: 970-491-7907; E-mail:kbeam{at}lamar.colostate.edu.
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Abstract |
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The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca2+ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:28092816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca2+ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca2+ currents at a few potentials (from -20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca2+ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca2+ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: act = 30.7 ± 1.9 ms, n = 11; CEIIIK:
act = 2.9 ± 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca2+ transients with parameters of voltage dependence (V0.5 = 6.5 ± 3.2 mV and k = 9.3 ± 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca2+ transients at any potential. Thus, high Ca2+ permeation is required for cardiac-type excitationcontraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to -50 mV (Qoff) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Qoff was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 ± 2.0%, n = 6). The increase in Qoff for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Qoff may reflect a voltage sensor movement required for activation of L-type Ca2+ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.
Key Words: voltage-dependant calcium channels, skeletal muscle, calcium transients, charge movement, ion channel
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Introduction |
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Skeletal and cardiac muscle dihydropyridine receptors (DHPRs)1 are highly homologous proteins that function both as voltage-gated L-type Ca2+ channels (L-channels) and as links between sarcolemmal depolarization and the release of Ca2+ from the sarcoplasmic reticulum (SR). Nonetheless, there are important differences between the two proteins. Cardiac L-channels activate 10-fold more rapidly upon depolarization (
In spite of these distinct functional properties, the selectivity of both skeletal (1 subunit (
Here, we have used expression in dysgenic myotubes to compare permeation and EC coupling in skeletal and cardiac L-channels after substitution of lysine for the repeat III glutamate. Compared with the wild-type channel, the mutant cardiac L-channel (CEIIIK) conducted small inward currents carried by Ca2+ and large outward currents carried by monovalent cations. The mutant skeletal L-channel (SkEIIIK) conducted large outward currents, but no detectable inward Ca2+ current. As a result of the greatly reduced Ca2+ entry, CEIIIK lost the ability of wild-type cardiac L-channels to trigger the release of SR Ca2+ in dysgenic myotubes. By contrast, SkEIIIK channels were able, despite the complete loss of Ca2+ entry, to trigger the release of SR Ca2+ with a voltage dependence similar to that of the wild type skeletal DHPR.
The production of large outward currents by the SkEIIIK and CEIIIK channels allowed us to compare activation rates over a much broader range of voltages than wild type channels, which can only be compared for test potentials that are both sufficiently positive to cause activation and sufficiently negative to provide a significant driving force for inward Ca2+ current. We found that the activation rate of both channels was very weakly voltage dependent and that the activation of SkEIIIK was >10-fold slower than that of CEIIIK, even at +100 mV.
Skeletal L-channels are unusual in that channel activation is both slow (act > 50 ms;
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Methods |
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Preparation of Dysgenic Myotubes
Primary cultures of myotubes were prepared from skeletal muscle of newborn dysgenic mice, as described previously (
Preparation and Expression of cDNAs
A glutamate-to-lysine mutation in the repeat III pore region of the skeletal muscle dihydropyridine receptor was constructed using PCR. Two initial PCR fragments were produced in separate but parallel reactions. Each initial fragment was generated by amplifying pCAC6 (
Approximately 1 wk after plating, myotubes were microinjected (
Measurements of Ionic Currents
The whole cell variant of the patch clamp technique ( when filled with the internal solution. Linear capacitative and leakage currents were determined by averaging the currents elicited by multiple (usually 10) 20-mV hyperpolarizing pulses from a holding potential of -80 mV. This control current was then scaled appropriately and used to correct test currents for linear components of capacitative and leakage currents. Electronic compensation was used to reduce the effective series resistance (usually to ~1 M
) and the time constant for charging the linear cell capacitance to <0.5 ms. Cell capacitance was determined by integration of the capacity transient resulting from the control pulse and was used to normalize currents (pA/pF) or charge movements (nC/µF) obtained from different myotubes. Ionic currents were filtered at 2 kHz and digitized at 1 kHz. To measure macroscopic L-current in isolation, a 1-s prepulse to -30 mV followed by a 25-ms repolarization to -50 mV was administered before the test pulse (prepulse protocol) to inactivate T-type Ca2+ currents.
The activation phase of L-currents was fitted by the following exponential function:
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(1) |
where I(t) is the current at time t after the depolarization, I is the steady state current, and
act is the time constant of activation. The voltage dependence of SkEIIIK L-channel activation was obtained by tail-current analysis. In brief, myotubes were stepped for 200 ms to test potentials (V) ranging from -50 to +80 mV; the instantaneous current (IT) was then measured immediately after stepping to +40 mV. These data were then normalized to Imax (the maximal IT) and fitted according to:
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(2) |
where VG is the potential causing half-maximal activation of L-type conductance and kG is a slope parameter.
Measurements of Gating Currents
For measurement of intramembrane charge movements, filtering was at 2 kHz (eight pole Bessel filter; Frequency Devices Inc.) and digitization was at 10 kHz. Voltage clamp command pulses were exponentially rounded with a time constant of 50300 µs and the prepulse protocol (see above) was used to reduce the contribution of gating currents from sodium channels and T-type Ca2+ channels. "OFF" transients of charge movement were measured for repolarization to -50 mV after test pulses to various test potentials (-50 to +60 mV, in 10-mV increments). The rectification of the mutant L-channels meant that influx of Ca2+ ions was minimal for CEIIIK and nonexistent for SkEIIIK (see Figure 1 and Figure 5). However, since some dysgenic myotubes exhibit a small, endogenous, rapidly activating L-current, residual Ca2+ channel ionic currents were blocked by the addition of 2.0 mM CdCl2 + 0.2 mM LaCl3 to the extracellular recording solution (see Solutions). The integral of the OFF transient for each test potential (V) was normalized by the maximal value of Qoff (Qmax) and fitted according to:
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(3) |
where VQ is the potential causing movement of half the maximal charge, and kQ is a slope parameter.
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Measurements of Intracellular Ca2+ Transients
Changes in intracellular Ca2+ were recorded with Fluo-3, as described previously (F/Fb, where
F represents the change in fluorescence from baseline (
F = F - Fb) and Fb represents the average myotube fluorescence immediately before depolarization.
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. For the measurements of depolarization-induced Ca2+ transients (
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Results |
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A Glutamate-to-Lysine Mutation in the Pore Region of Repeat III Alters L-Channel Permeability, but Not the Rate of Channel Activation
Figure 1 shows ionic currents in dysgenic myotubes expressing SkEIIIK and CEIIIK, the skeletal and cardiac DHPRs, respectively, in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Under the recording conditions used, the wild-type skeletal and cardiac L-channels support inward Ca2+ currents for test potentials ranging from about -20 (cardiac) or 0 (skeletal) mV to +60 mV or greater, with the currents reversing to outward at an extrapolated potential of more than +70 mV (
Figure 1 B illustrates peak currentvoltage relationships determined from the cells illustrated in Figure 1 A. Compared with wild-type cardiac channels, the reversal potential for CEIIIK was negatively shifted by ~60 mV, indicative of a large change in the permeability of Ca2+ relative to Cs+. The negative shift in reversal potential appeared to have been still larger for SkEIIIK since inward Ca2+ currents were not elicited at any test potential, even though depolarizations >10 mV caused significant activation of SkEIIIK channels (see Figure 2 B). Furthermore, when outward currents via SkEIIIK were elicited by strong depolarizations, subsequent repolarization to negative potentials failed to cause inward ionic tail currents (see below). Thus, the conserved glutamate in the pore region of repeat III appears to play a role in controlling Ca2+ permeation through the skeletal L-channel, which is even more pivotal than its role in the cardiac L-channel.
Like wild-type skeletal L-channels, the SkEIIIK channels activated slowly. At test potentials where both produce appreciable currents (e.g., +60 mV), the SkEIIIK and native skeletal L-channels both display slow activation (native: 52.4 ± 2.0 ms, n = 6; SkEIIIK: 40.8 ± 1.7 ms, n = 11). Thus, the identity of the permeating ion has little effect on activation and the EIIIK channels provide a convenient tool for extending the comparison of skeletal and cardiac activation to more positive potentials (Figure 1 C). Even at +100 mV, activation of SkEIIIK is more than 10-fold slower than that of CEIIIK (SkEIIIK: 30.7 ± 1.9 ms, n = 11; CEIIIK: 2.9 ± 0.5 ms, n = 7). Because activation of the skeletal L-channel is slow over a broad range of test potentials (+20 to +100 mV), the rate-limiting event for the transition of the channel from closed to open must be very weakly voltage dependent, as we have suggested previously from single channel analyses (
L-Channel Permeability to Ca2+ Is Required for Cardiac-type, but Not Skeletal-type, EC Coupling
Controversy remains as to whether Ca2+ permeation and/or intrapore Ca2+ binding are necessary for the ability of the cardiac and skeletal L-channels to mediate EC coupling. We reexamined this issue with the CEIIIK and SkEIIIK L-channels since they have a profoundly altered intrapore binding site for Ca2+. In initial experiments, contractions were observed in response to extracellular electrical stimulation (80 V, 1030 ms) of dysgenic myotubes injected with cDNA for SkEIIIK but not in myotubes injected with CEIIIK cDNA. Thus, the substantial loss of Ca2+ permeation caused by the EIIIK mutation appeared to have eliminated the ability of the cardiac L-channel, but not the skeletal L-channel, to mediate EC coupling. However, to determine whether CEIIIK supports Ca2+ release too small to be detected by contractions, we also used whole-cell voltage clamping to measure membrane currents and intracellular Ca2+ transients with patch pipettes containing the pentapotassium salt of Fluo-3 (
Figure 2 A illustrates Ca2+ transients and membrane currents in dysgenic myotubes expressing either SkEIIIK (left) or CEIIIK (right). Large Ca2+ transients were present in the myotube expressing SkEIIIK (left), but not in the one expressing CEIIIK, despite the presence of large ionic currents (right). Similar results were obtained in five myotubes expressing SkEIIIK, all of which displayed depolarization-evoked Ca2+ transients, and five myotubes expressing CEIIIK, none of which displayed Ca2+ transients. These data lend strong support to the notion that Ca2+ permeation through skeletal muscle DHPRs is not required to trigger the release of Ca2+ from the SR (
Figure 2 B plots both normalized conductance (G) and the amplitude of the Ca2+ transient (F/F) as a function of test potential for SkEIIIK-expressing myotubes. As in myotubes expressing the wild-type skeletal DHPR (
F/F (VF = 6.5 ± 3.2 mV, kF = 9.3 ± 0.7 mV, n = 5) for SkEIIIK L-channels are also similar to the values reported previously (
An Increase in Qoff Correlates with Slow Activation of Skeletal L-Current
In comparison with other voltage-gated Ca2+ channels, skeletal L-channels have extremely slow (and relatively voltage-independent) activation kinetics. Despite this slow activation, repolarization to negative potentials causes rapid deactivation, a behavior that can be explained by a single transition, with an asymmetric voltage dependence causing it to be slow in the opening direction and fast in the closing direction (
Figure 3 illustrates representative Qoff gating currents generated upon repolarization to -50 mV for dysgenic myotubes expressing either SkEIIIK (left) or CEIIIK (right). Data were obtained in the presence of 2.0 mM Cd2+ and 0.2 mM La3+ (which was required to block the small inward currents supported by CEIIIK). Qoff is shown after either brief (20 ms, light traces) or long (200 ms, dark traces) depolarizing pulses to the indicated potentials. These two pulse durations were used because 20 ms is long enough to open cardiac L-channels fully and short enough to only marginally open skeletal L-channels, whereas 200 ms is sufficiently long to open both cardiac and skeletal L-channels. At potentials less than +10 mV, Qoff was independent of the test pulse duration for both SkEIIIK and CEIIIK. However, at potentials that typically activate ionic current (greater than or equal to +10 mV), Qoff was significantly larger only for SkEIIIK L-channels after the 200-ms test pulses. The additional Qoff for SkEIIIK L-channels was manifested as a prolongation of the total gating current rather than simply as an increase in peak of the "OFF" gating current. Thus, the increase in SkEIIIK Qoff caused by longer depolarizations is inconsistent with a contamination from an ionic tail current and presumably arises from an additional amount of gating current recruited during slow channel activation. In contrast to SkEIIIK, the magnitude of Qoff for CEIIIK was not increased by the longer test pulse duration. Rather, a slight decrease in Qoff was found for CEIIIK after the largest 200-ms depolarizations (to potentials greater than +30 mV), most likely owing to a small degree of inactivation during the 200-ms pulses.
Figure 4 illustrates the voltage dependence of Qoff for SkEIIIK (A) and CEIIIK (B) after 20-ms test pulses (Q20; ) and 200-ms test pules (Q200; ), and the difference between the two (Q200Q20;
). For potentials >0 mV, Qoff SkEIIIK was larger after 200-ms test pulses than after 20-ms test pulses. For CEIIIK, no such increase in Qoff occurred after longer duration pulses. The normalized voltage dependence of Q20, Q200, Q200Q20, and channel conductance for the SkEIIIK-expressing myotube shown in Figure 3 are compared in Figure 4 C. As reported previously for Qon (
) exhibited a voltage dependence nearly identical to that of channel conductance (
). In a total of four experiments, good agreement was found between the voltage dependence of conductance (VG = 24.2 ± 6.3 mV and kG = 7.5 ± 1.0 mV) and that of Q200Q20 (VQ = 31.4 ± 6.7 mV and kQ = 7.5 ± 0.7 mV). Figure 4 D summarizes the results on maximal Qoff obtained from multiple experiments. On average, the maximal value of Qoff for SkEIIIK after 200-ms test pulses was significantly greater (121.4 ± 2.0%, n = 6, P < 0.001) than that after 20-ms test pulses. For CEIIIK, Qoff after 200 ms was not significantly different from that after 20 ms (P > 0.3). Additionally, for both SkEIIIK and CEIIIK, Qoff after 20 ms had essentially the same magnitude as Qon (data not shown). Thus, the voltage and time dependence of the Q200Q20 gating charge, coupled with its presence only in the SkEIIIK channels, suggests that this extra gating charge underlies the rate-limiting transition of slow skeletal channel activation.
Since contamination by inward ionic tail current would artifactually increase Qoff, it was important to ensure that no such contamination was present under our recording conditions. As one approach, we attempted to reduce ionic currents by adding Cd2+ and La3+ to the external bath. At a test potential of +60 mV, these blockers reduced the outward current by 75.3 ± 2.6% (n = 5) for SkEIIIK and 82.0 ± 2.7 (n = 5) for CEIIIK. To determine the effectiveness of this block at more negative potentials, cells were depolarized to +60 mV for 200 ms to cause maximal activation of channels and then either maintained at +60 mV or repolarized to varying potentials. As shown for a SkEIIIK-expressing cell in Figure 5, the addition of Cd2+ and La3+ caused a large reduction at +60 mV, where the integrated current was entirely ionic, but only a small reduction at more negative potentials. The small effect of the blockers at negative potentials strengthens the conclusion that SkEIIIK has a very low permeability to Ca2+. Moreover, it seems likely that the block of this already small Ca2+ permeability by Cd2+ and La3+ should have been sufficient to eliminate any ionic contamination of the Qoff charge.
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Discussion |
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Mutation of Glutamate to Lysine in the Pore Region of Repeat III Eliminates Ca2+ Permeation through the Skeletal L-Type Ca2+ Channel
In this paper, we have characterized the effects of mutational alteration of Ca2+ permeability on the behavior of skeletal and cardiac L-channels expressed in dysgenic myotubes. Because there had been no previous descriptions of mutations affecting Ca2+ permeability of skeletal L-channels, we chose to substitute lysine for glutamate in the repeat III pore region (EIIIK) since the corresponding mutation had already been shown to produce a dramatic alteration in the divalent selectivity of the cardiac L-channel expressed in Xenopus oocytes (
Reconstitution of EC Coupling in Dysgenic Myotubes: Ca2+ Permeation and Skeletal and Cardiac EC Coupling
Previous studies have used expression in dysgenic myotubes of skeletal, cardiac, and chimeric DHPRs to dissect mechanisms of EC coupling. With this approach, it was shown that substitution of skeletal sequence for all (
Occupancy of a metal cation binding site, which is accessible from the extracellular space, has been reported to be required for the conformational changes of the skeletal L-channel that result in the activation of RyR1s (
The results with SkEIIIK raise the obvious question of what function is played by the slowly activating L-type Ca2+ current in skeletal muscle. Clearly, this current plays little or no role in contractions elicited by relatively brief depolarizations. Perhaps the Ca2+ current becomes important for contraction only during tetanic stimulation (
Gating Charge Associated with Slow Activation of Skeletal L-Current
As in the case for inward Ca2+ currents via wild-type skeletal L-channels, outward currents carried by SkEIIIK L-channels also activate slowly. Moreover, because they are slow over a broad range of test potentials, the SkEIIIK L-currents extend previous conclusions that activation of skeletal L-channels is only very weakly voltage dependent (Figure 1 C). Nonetheless, steady state activation is strongly voltage dependent for wild-type ( 8 mV, which corresponds to an effective valence of approximately three electronic charges. This effective valence represents ~25% of the total gating charge expected for voltage-gated calcium channels (
A simple linear kinetic scheme consistent with the second possibility above, as well as with measurements of unitary currents through skeletal L-channels (
In this model, rapid gating currents are produced by closedclosed transitions, which for simplicity are indicated by the single C0C1 transition. These gating transitions could be important for triggering release of Ca2+, since Ca2+ release has a voltage dependence much closer to that of Qon than does L-channel conductance ( and
, respectively. The rate constant
must be small and not strongly voltage dependent because activation remains slow for SkEIIIK even at +100 mV. By contrast, the rate constant
must be strongly voltage dependent such that it is small at the depolarized potentials causing activation of current and large at negative voltages (e.g., -50 mV) causing deactivation. This behavior, which suggests that the energy barrier separating C1 and C2 is asymmetrically located with respect to the transmembrane potential drop, is required because activation is slow, whereas deactivation is rapid (
Based on chimeras of the skeletal and cardiac L-channels, it was shown that the sequence of IS3 and the IS3IS4 linker determines whether activation is fast or slow (
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Footnotes |
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Portions of this work have been previously published in abstract form (Dirksen, R.T., and K.G. Beam. 1996. Biophys. J. 70:A146).
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Acknowledgements |
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We thank Dr. William Sather for providing us with the cDNA for CEIIIK. We also thank Robin Morris, Kim Lopez-Jones, and Katherine Parsons for expert technical assistance.
This study was supported by the Muscular Dystrophy Association (to R.T. Dirksen) and National Institutes of Health grants NS-24444, AR-44750 (to K.G. Beam), and AR44657 (to R.T. Dirksen).
Submitted: May 7, 1999; Revised: June 24, 1999; Accepted: June 29, 1999.
1used in this paper: DHPR, dihydropyridine receptor; EC, excitationcontraction; L-channel, L-type Ca2+ channel; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; TEA, tetraethylammonium
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References |
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