Correspondence to: Stephen C. Cannon, EDR 413 / Massachusetts General Hospital, Fruit Street, Boston, MA 02114. Fax:617-726-3926 E-mail:cannon{at}helix.mgh.harvard.edu.
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Abstract |
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Skeletal muscle dihydropyridine (DHP) receptors function both as voltage-activated Ca2+ channels and as voltage sensors for coupling membrane depolarization to release of Ca2+ from the sarcoplasmic reticulum. In skeletal muscle, the principal or 1S subunit occurs in full-length (
10% of total) and post-transcriptionally truncated (
90%) forms, which has raised the possibility that the two functional roles are subserved by DHP receptors comprised of different sized
1S subunits. We tested the functional properties of each form by injecting oocytes with cRNAs coding for full-length (
1S) or truncated (
1S
C)
subunits. Both translation products were expressed in the membrane, as evidenced by increases in the gating charge (Qmax 80150 pC). Thus, oocytes provide a robust expression system for the study of gating charge movement in
1S, unencumbered by contributions from other voltage-gated channels or the complexities of the transverse tubules. As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba2+ currents were small relative to Qmax. The truncated
1S
C protein, however, supported much larger ionic currents than the full-length product. These data raise the possibility that DHP receptors containing the more abundant, truncated form of the
1S subunit conduct the majority of the L-type Ca2+ current in skeletal muscle. Our data also suggest that the carboxyl terminus of the
1S subunit modulates the coupling between charge movement and channel opening.
Key Words: Ca2+ channels, skeletal muscle, Xenopus oocyte expression, cut-open oocyte voltage clamp, gating charge movement
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INTRODUCTION |
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Skeletal muscle dihydropyridine (DHP)1 receptors are L-type Ca2+ channels that serve a dual role in muscle, functioning both as Ca2+-conducting pores and as the voltage sensors for excitationcontraction (E-C) coupling (1S subunit plus the ß1,
2
, and
regulatory subunits (
1S subunit have been detected in skeletal muscle: a 212-kD full-length translation product (
1S), present as a minor component, and a more abundant 175-kD form (
1S
C, 90% of
1 subunit recovered from transverse tubule membrane preparations) created by post-translational cleavage of 175 amino acids from the COOH terminus (
1 subunits have been shown to play a role in channel regulation (
1S is unknown.
One long-standing hypothesis holds that some DHP receptors in skeletal muscle are specialized voltage sensors for E-C coupling, while others are capable of both voltage sensing and Ca2+ conduction. Comparison of DHP binding to L-type Ca2+ current amplitude in intact muscle fibers suggested that only a small percentage of DHP receptors are functional Ca2+ channels (1S occurring in a 90%:10% ratio led to the hypothesis that the rarer full-length form is capable of both sensing the voltage and passing current, while the more abundant truncated form is a dedicated voltage sensor for E-C coupling (
Injection of cDNAs encoding either 1S or a COOH-terminally truncated form of
1S (at Asn 1662) into the nuclei of dysgenic mouse myotubes lacking
1S restores contraction, gating charge movement, and L-type Ca2+ current (
1S
C used in these experiments was artificially truncated slightly upstream of the native cleavage site, these results clearly suggest that
1S
C can function as both an ion channel and a voltage sensor for E-C coupling. However, the role of full-length
1S remains unknown, since it is possible that an undetermined proportion of the
1S subunits were cleaved to
1S
C in the myotubes. One recent study has address this issue. When the green fluorescent protein was fused to the COOH terminus of
1S and expressed in dysgenic myotubes, the fluorescence was localized to the t tubules, which suggests a significant amount of full-length
1S was inserted into the membrane and little cleavage occurred within the 4872-h duration of the experiment (
Heterologous expression in nonmuscle cells provides an alternative opportunity to distinguish the functional roles of the two 1S forms. Injection of cRNAs encoding
1S
C plus the muscle-associated ß1b,
2
, and
subunits into Xenopus oocytes was recently found to give rise to robust DHP-sensitive L-type currents, whereas injection of full-length
1S cDNA gave little or no current (
1S and
1S
C, they leave open the possibility that full-length
1S simply expresses poorly in oocytes, but conducts Ca2+ just as well as
1S
C. Moreover, measurements of ionic current alone cannot address the crucial question of whether
1S and
1S
C function differently as voltage sensors.
To study the current-carrying and voltage-sensing capabilities of the two size forms of 1S in parallel, we expressed
1S and
1S
C with the ß1,
2
, and
auxiliary subunits in Xenopus oocytes. We examined the ß1a and ß1b splice variants, both of which are expressed in muscle tissue (
1S subunit supported gating charge movements in 2 mM Co2+ when expressed with auxiliary subunits in oocytes, even though only the truncated form conducted appreciable L-type currents in 10 mM Ba2+. This result was independent of whether the ß1a or ß1b splice variant was used. We conclude that, while
1S and
1S
C function almost identically as voltage sensors,
1S
C is the form specialized to carry L-type current, while
1S appears to be somehow inhibited from passing substantial ionic current.
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METHODS |
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The rabbit 1S, rat brain ß1b, rabbit
2
, and rabbit
Ca2+ channel cDNAs were obtained in the pKCRH, pBluescript, pcDNA3, and pcD-x vectors, respectively, as a gift from Dr. Kevin Campbell (University of Iowa, Iowa City, IA). The rabbit ß1a subunit was obtained in the pNKS2 vector as a gift from Dr. Bernhard Flucher (University of Innsbruck, Innsbruck, Austria).
1S and
were subcloned into the pGEMHE oocyte expression vector (gift of Dr. Emily Liman, Harvard University) at the HindIII and BamHI polylinker sites, respectively. ß1b was subcloned into pGEMHE between the SacII and HindIII sites, while
2
and ß1a were left in pCDNA3 and pNKS2, respectively.
1S
C-pGEMHE was made from
1S-pGEMHE using the Clontech Transformer site-directed mutagenesis kit (CLONTECH Laboratories, Inc.). The mutagenic primer caused a loop-out deletion of base pairs 53205844 (amino acids 16981893) of the rabbit
1S sequence, while the selection primer mutated the MfeI site at position 2788 of the pGEMHE sequence to a unique NdeI site. For in vitro synthesis of RNA, the plasmids containing the Ca2+ channel subunits were linearized using the following restriction enzymes: Sse8387I for
1S and
1S
C, XbaI for ß1a, NotI for ß1b, PvuII for
2
, and NheI for
. Capped cRNA was synthesized using the mMessage mMachine T7 kit (for
1S,
1S
C, ß1b,
2
, and
) or SP6 kit (for ß1a; Ambion Corp.) and extracted using the RNAid purification kit (Bio101).
Stage V and VI oocytes were harvested from egg-bearing female Xenopus laevis frogs under anesthesia with 3-aminobenzoic acid ethyl ester (1 mg ml-1 in a cold water bath for 25 min; Sigma-Aldrich), in accordance with the guidelines of the Subcommittee on Research Animal Care at the Massachusetts General Hospital. Oocytes were removed into Ca2+-free OR-2 solution containing (mM): 82.5 NaCl, 2.5 KCl, 1 MgCl2, and 5 HEPES, pH 7.6. The egg sacs were manually torn open using forceps, and the oocytes were incubated in OR-2 containing 2 mg ml-1 collagenase (GIBCO BRL) for 2.5 h in a room temperature shaker (60 rpm) to remove the follicular membrane. The oocytes were then washed four times in OR-2 solution and transferred for storage to ND-96 solution containing (mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 2.5 pyruvate, 5 HEPES, and 50 µg ml-1 gentamicin (GIBCO BRL), pH 7.6. Oocytes were injected with 50100 nl of a 1:1:1:1 mixture of 1S (or
1S
C), ß1a (or ß1b),
2
, and
cRNA using injection pipettes pulled from thin-wall capillary glass (World Precision Instruments) and were stored at 18°C for 57 d before recording.
Oocytes were voltage clamped in the cut-open configuration, with active clamp of the upper and guard compartments, using an oocyte clamp (CA-1B; Dagan Corp.) under control of a custom stimulation/recording program written in AxoBASIC and running on an IBM-compatible computer. Voltage-sensing electrodes, fabricated from borosilicate capillary glass (1.65-mm outer diameter; VWR Scientific) using a multi-stage puller (Sutter Instrument Co.), were filled with 3 M KCl and had resistances of 0.21 M. The leads of the amplifier headstage, attached to Ag/AgCl pellets in plastic wells containing 2 M NaCl solution, were connected to the upper, guard, and lower oocyte compartments using glass agar bridges containing 110 mM Na-methanesulfonic acid and 10 mM HEPES, in 3% agarose, pH 7.0, and threaded with a platinum wire. Current traces, evoked by depolarizing test pulses from a holding potential of -90 mV, were corrected for linear leak and capacity currents (minimized by the analog compensation of the amplifier) by addition of control currents evoked by hyperpolarizing pulses of equal amplitude from the holding potential (P/-1 protocol). Since the charge movement was not noticeably altered by changing the holding potential to -110 or -120 mV (data not shown), we judged our leak subtraction protocol to be sufficient to selectively subtract linear components. Signals were filtered at 1 kHz and sampled at 20 kHz (for gating currents) or 2 kHz (for ionic currents). Analysis and curve fitting was performed using a combination of custom AxoBASIC routines and SigmaPlot (Jandel Scientific). Student's t test was used to make pairwise comparisons between sets of data (P values are reported in the text; P = 0.05 was taken as the limit of statistical significance).
Solutions for recording Ba2+ currents and Ca2+ channel gating currents were prepared following
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RESULTS |
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Oocytes injected with either 1S or
1S
C plus the auxiliary subunit cRNAs showed transient currents at the beginning and end of short depolarizing pulses, while uninjected oocytes and oocytes injected with auxiliary subunit cRNAs alone lacked these currents (Fig 1 A). These transient currents had the properties expected of gating currents arising from the movement of charged elements in the ion channel protein: (a) they depended on the presence of the pore-forming
subunit (Fig 1 A); (b) they always followed the direction of the voltage step (A); (c) the integral of the current at the onset of a voltage step (Qon) matched the integral of the current at the offset of the step (Qoff) (B); and (d) Qon and Qoff approached limiting values at both positive and negative potentials, with a voltage dependence that followed a Boltzmann distribution (C).
|
Fig 2, which compares the magnitude and voltage dependence of the charge movement measured in oocytes expressing 1S and
1S
C, shows that both constructs were clearly expressed abundantly at the surface membrane regardless of which ß1 subunit isoform was coexpressed. Qoff was used for the comparison instead of Qon because of the faster kinetics (and hence greater single-to-noise ratio at small test depolarizations) of the OFF response. The maximum Qoff appeared larger in oocytes expressing
1S
C than in oocytes expressing
1S (Fig 2A and Fig B). While this effect on Qoff, max was not statistically significant in the presence of ß1a (Fig 2 A; P = 0.13), it was statistically significant in the presence of ß1b (B; P = 0.008). The normalized Qoff versus voltage (Q-V) curves for
1S and
1S
C had roughly the same midpoints and slopes (Fig 2 C). As has been observed for other voltage-dependent ion channels and for native L-type Ca2+ channels in skeletal muscle, the Q-V curve started at significantly more negative potentials and was less steep than the G-V curve for L-type current (Fig 2 C,
). This is consistent with the idea that skeletal muscle Ca2+ channels undergo rapid voltage-dependent transitions among several closed conformations before opening, some of which are presumably involved in excitationcontraction coupling (
|
To compare directly the voltage-sensing and current-carrying properties of 1S and
1S
C, we measured both ionic and gating currents in individual oocytes expressing these constructs (Fig 3). In these experiments, ionic currents were measured in the presence of 10 mM Ba2+, the extracellular Ba2+ was then replaced with 2 mM Co2+ using a manual pipet, and gating charge movements were measured (see Fig 3, legend). For these experiments, Qon was measured instead of Qoff, despite the lower signal-to-noise ratio of the ON response, to avoid the possibility of contamination by small Ba2+ tail currents remaining after the solution exchange. These experiments confirmed that the full-length form of the channel conducts Ba2+ current very poorly compared with the truncated form (
1S resulted in large voltage-driven gating charge movements not found in uninjected eggs. Moreover, this difference was observed whether ß1a (Fig 3A and Fig B) or ß1b (C and D) cRNA was coinjected. The maximal amplitude of the gating currents measured in each egg was comparable with that of the largest ionic current, consistent with the idea that channel expression was high but the channel open probability was low. Indeed, while the ionic currents, even those carried by
1S
C, were small compared with those measured for other cloned Ca2+ channels expressed in oocytes, the gating currents were as large as or larger than those of other Ca2+ channels (
1S to be mostly, though not completely, endogenous in origin (a conclusion confirmed by separate experiments in which 5 µM nimodipine was found to block <10% of the current expressed in these cells; data not shown). These currents were consistently smaller than the endogenous current observed in oocytes injected with the auxiliary subunit cRNAs alone, most likely because of competition between
1S subunits and endogenous Ca2+ channel subunits for the auxiliary subunits.
|
Comparison of the mean Qon-V and I-V curves measured in oocytes expressing 1S
C and
1S with either the ß1a subunit (Fig 4A and Fig B) or the ß1b subunit (C and D) emphasizes that while the full-length and truncated subunits are comparable voltage sensors, the truncated version is a much better conductor of ionic current. We quantified this functional distinction by calculating the ratio of maximum ionic current to maximum charge movement (Imax/Qon, max) for each oocyte (Fig 4 E). Imax/Qon, max was sixfold greater for oocytes expressing
1S
C than for oocytes expressing
1S when either the ß1a or the ß1b subunit was used; a difference that was highly statistically significant in each case (P = 4.8 x 10-6 and 6.2 x 10-5, respectively). This difference is a conservative estimate, since at least 90% of the current observed in
1S-injected oocytes was conducted by a DHP-insensitive
subunit, as mentioned above (data not shown). Imax/Qon, max was smaller for channels containing ß1a than for channels containing ß1b, reflecting both the somewhat smaller ionic currents and larger gating currents observed, on average, when ß1a was injected. As was also seen in the separate experiments of Fig 2, truncation of the
1S increased the size of gating currents significantly in the presence of the ß1b subunit (P = 0.02), but not in the presence of the ß1a subunit (P = 0.63).
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DISCUSSION |
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The primary goal of this study was to determine whether the different magnitudes of L-type Ba2+ current observed previously when the full-length 1S and truncated
1S
C subunits were expressed in Xenopus oocytes (
1S or
1S
C with the ß1 (ß1a or ß1b),
2
, and
subunits that represented gating charge movements in the
1 subunit molecule, demonstrating that both size forms of the channel are expressed in oocytes and constitute working voltage sensors. The range and steepness of the voltage dependence of the charge movement mediated by
1S and
1S
C were essentially the same, independent of which ß1 subunit was coexpressed. The maximum amount of charge moved at depolarized voltages, Qmax, tended to be larger for
1S
C than for
1S. However, this difference in Qmax was statistically significant (both Qon, max and Qoff, max) only when the ß1b subunit, rather than the ß1a subunit, was coexpressed. Measurements of both charge movement and ionic current from the same oocyte showed that: (a) oocytes expressing the truncated subunit plus auxiliary subunits supported both charge movements and L-type Ba2+ current; (b) oocytes expressing the full-length subunit plus auxiliary subunits had charge movements but very little L-type Ba2+ current; and (c) oocytes expressing the auxiliary subunits alone had neither detectable charge movements nor L-type Ba2+ current. The stark differences observed between cells expressing
1S versus
1S
C suggest there was no appreciable COOH-terminal truncation of
1S in the oocyte.
We conclude that the two naturally occurring size forms of the skeletal muscle DHP receptor subunit are functionally specialized when expressed with the auxiliary subunits found in muscle.
1S and
1S
C are both functional voltage sensors, but
1S
C is a much more effective Ca2+ channel. Only tiny ionic currents were seen when the
1S subunit was coexpressed with either the ß1a or ß1b subunit (Fig 4A and Fig C), and the currents observed when the
1S/ß1b/
2
/
combination was injected were
90% insensitive to 5 µM nimodipine (data not shown). This differs from the data of
1S was expressed with ß1b,
2
, and
(although this conclusion was based on current kinetics rather than on dihydropyridine block). We observed robust ionic currents when either ß1a or ß1b cRNAs were coinjected with
1S
C cRNA, in contrast to the original report of a requirement for ß1b (
1S
C/ß1a/
2
/
combination confirmed that the inward current seen in this case was 8090% L-type (data not shown). Our results therefore support the hypothesis that the predominant form of native DHP receptors in skeletal muscle, those containing
1S
C and ß1a, can function both as voltage sensors and as L-type calcium channels. Given the strong similarity of the kinetics and voltage dependence of the ionic and gating currents measured here in oocytes to those measured previously in mammalian muscle cells (
1S and
1S
C in oocytes are likely to occur in native DHP receptors. Nevertheless, experimental confirmation of the distinct functional roles of the
1S and
1S
C subunits must ultimately be delineated in native cells, especially given the mounting evidence from dyspedic myotubes that both functions of the DHP receptor are strongly influenced by interacting ryanodine receptors (
1S subunit may ultimately require the identification and selective inhibition of the enzyme responsible for COOH-terminal truncation.
The mechanism by which the carboxyl terminus modulates the coupling between voltage-dependent gating and channel opening remains unknown. A similar effect has been observed for the cardiac L-type Ca2+ channel. Deletion of amino acids 307472 from the 665-amino acid carboxyl terminus of the 1C subunit increased Ba2+ currents four- to sixfold in the oocyte expression system (
subunits. The carboxyl terminus deletion of
1S studied herein removes several consensus sites for PKC and cAMP-mediated phosphorylation, but leaves intact an EF hand and calmodulin-binding IQ consensus motifs, both of which have been implicated in Ca2+-dependent modulation of inactivation and facilitation of ionic current (
1S
C is dependent on any of these previously identified modulatory roles of the carboxyl terminus. Our observation that truncation of the
1S subunit significantly increased the magnitude of gating charge movements (an effect not seen with COOH-terminal truncation of
1C) suggests that the COOH terminus of
1S may play a role in channel expression in the membrane as well as in the coupling of gating to pore opening.
Our experiments give the unexpected result that both size forms of the 1S subunit express as well as or better than other cloned Ca2+ channel isoforms in Xenopus oocytes, as judged by gating current amplitudes (
1S in heterologous systems (
1S subunit. Robust expression of the skeletal muscle L-type Ca2+ channel in a heterologous system will be useful in elucidating the functional consequences of muscle-specific modulatory mechanisms, which may include modifications to the channel protein (such as phosphorylation or the COOH-terminal truncation studied here) or interactions with other proteins (such as the auxiliary Ca2+ channel subunits or other components of the triad junction). In addition, the ability to record skeletal muscle Ca2+ channel gating currents in an expression system lacking the electrically complex features of muscle cells, such as t tubules and additional voltage-gated channels, may allow for unprecedented precision in the study of the channel voltage sensor.
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Footnotes |
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1 Abbreviations used in this paper: DHP, dihydropyridine; E-C, excitationcontraction.
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Acknowledgements |
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The authors thank B.P. Bean for lending the oocyte clamp and for comments on the manuscript.
This work was supported by the National Institute of Arthritis, Musculoskeletal and Skin Diseases (AR42703 to S.C. Cannon) and the Quan Fellowship to Harvard Medical School (J.A. Morrill).
Submitted: 20 April 2000
Revised: 5 July 2000
Accepted: 18 July 2000
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