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Effects of mutations causing hypokalaemic periodic paralysis on the skeletal muscle L-type Ca²⁺ channel expressed in Xenopus laevis oocytes

James A. Morrill * and Stephen C. Cannon *¹²

MS 9476 Received 6 April 1999; accepted after revision 3 August 1999.

	ABSTRACT

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1. A truncated form of the rabbit _1S Ca²⁺ channel subunit (_1SC) was expressed with the _1b, ₂ and auxiliary subunits in Xenopus laevis oocytes. After 5-7 days, skeletal muscle L-type currents were measured (469 ± 48 nA in 10 mM Ba²⁺). All three of the auxiliary subunits were necessary to record significant L-type current. A rapidly inactivating, dihydropyridine-insensitive endogenous Ba²⁺ current was observed in oocytes expressing the auxiliary subunits without an exogenous subunit. Expression of full-length _1S gave 10-fold smaller currents than the truncated form.

2. Three missense mutations causing hypokalaemic periodic paralysis (R528H in domain II S4 of the _1S subunit; R1239H and R1239G in domain IV S4) were introduced into _1SC and expressed in oocytes. L-type current was separated from the endogenous current by nimodipine subtraction. All three of the mutations reduced L-type current amplitude (~40 % for R528H, ~60-70 % for R1239H and R1239G).

3. The disease mutations altered the activation properties of L-type current. R528H shifted the G(V) curve ~5 mV to the left and modestly reduced the voltage dependence of the activation time constant, _act. R1239H and R1239G shifted the G(V) curve ~5-10 mV to the right and dramatically slowed _act at depolarized test potentials.

4. The voltage dependence of steady-state inactivation was not significantly altered by any of the disease mutations.

5. Wild-type and mutant L-type currents were also measured in the presence of (-)-Bay K8644, which boosted the amplitude ~5- to 7-fold. The effects of the mutations on the position of the G(V) curve and the voltage dependence of _act were essentially the same as in the absence of agonist. Bay K-enhanced tail currents were slowed by R528H and accelerated by R1239H and R1239G.

6. We conclude that the domain IV mutations R1239H and R1239G have similar effects on the gating properties of the skeletal muscle L-type Ca²⁺ channel expressed in Xenopus oocytes, while the domain II mutation R528H has distinct effects. This result implies that the location of the substitutions is more important than their degree of conservation in determining their biophysical consequences.

	INTRODUCTION

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The skeletal muscle L-type Ca²⁺ channel plays a central role in muscle physiology, serving both as an ion-conducting pore and as the voltage sensor for excitation-contraction coupling (Tanabe et al. 1988; Melzer et al. 1995). Missense mutations in the gene encoding the pore-forming _1S subunit cause the dominantly inherited human muscle disorder hypokalaemic periodic paralysis (HypoPP), in which sporadic muscle paralysis occurs in conjunction with an acute reduction in the serum potassium concentration (Rüdel et al. 1984; Cannon, 1998). Three specific mutations have been identified in families with HypoPP: R528H, a substitution of histidine for the outermost arginine of domain II S4; R1239H, an equivalent substitution for the second arginine of domain IV S4; and R1239G, a substitution of glycine for the same residue (Fontaine et al. 1994; Jurkat-Rott et al. 1994; Ptacek et al. 1994; Fouad et al. 1997).

Because the HypoPP mutations affect conserved regions of the channel sequence thought to play a role in sensing voltage, several attempts have been made to determine their effects on channel gating (Sipos et al. 1995; Lapie et al. 1996; Lerche et al. 1996; Jurkat-Rott et al. 1998; Morrill et al. 1998). Recent studies of human myotubes cultured from heterozygous patients carrying the R528H mutation have given conflicting results. Jurkat-Rott et al. (1998) observed a 5 mV hyperpolarizing shift in the G(V) curve and the voltage dependence of steady-state inactivation without changes in the level of expression or the kinetics of activation or inactivation. In contrast, Morrill et al. (1998) found no effect of R528H on the G(V) curve or the voltage dependence of steady-state inactivation but observed a slowing of the rate of activation that became more pronounced with depolarization, as well as a mild reduction in L-type current density.

In order to define the biophysical effects of the HypoPP mutations more precisely, it would be desirable to compare all three of the mutations side by side in a well-controlled heterologous expression system. While the skeletal muscle L-type Ca²⁺ channel has been studied extensively in voltage-clamped mature muscle fibres (Cota et al. 1983; Sanchez & Stefani, 1983; Delbono, 1992; Francini et al. 1992; Garcia et al. 1992), cultured myotubes (Beam & Knudson, 1988; Rivet et al. 1992; Sipos et al. 1995, 1997; Jurkat-Rott et al. 1998; Morrill et al. 1998), and lipid bilayers fused with t-tubule membrane vesicles (Affolter & Coronado, 1985; Ma et al. 1991; Mejia-Alvarez et al. 1991), heterologous expression of cloned isoforms of the channel in more easily clamped cells, such as tsA-201 cells (Johnson et al. 1997), mouse L cells (Perez-Reyes et al. 1989; Lacerda et al. 1991; Varadi et al. 1991; Lapie et al. 1996) and Xenopus laevis oocytes (Dascal et al. 1992; Nargeot et al. 1992; Grabner et al. 1996), has proved difficult. The small currents observed when _1S is expressed in these cells alone or with various auxiliary subunits have suggested that the unique anatomical context of the channel - in the junctional t-tubule membrane, closely apposed to the foot processes of ryanodine receptors in the sarcoplasmic reticular membrane - may be crucial for its function. Expression of the cloned rabbit _1S subunit in dysgenic mouse myotubes by injecting cDNA into cell nuclei has provided one way to circumvent this problem (Tanabe et al. 1988,1991; Nakai et al. 1994; Garcia et al. 1997). Dysgenic myotubes injected with the rabbit _1S-R528H subunit were found to express L-type currents with gating properties very similar to wild-type (Jurkat-Rott et al. 1998).

Recently, Ren & Hall (1997) demonstrated significant expression of the skeletal muscle L-type Ca²⁺ channel in Xenopus oocytes when cRNAs encoding all four of the channel subunits (_1S, ₁, ₂ and ) were injected. Full expression (up to 3-4 µA per oocyte in 40 mM Ba²⁺ solution) was dependent on using a truncated version of _1S, which is the dominant form of the subunit in native muscle and is formed by post-translational cleavage near the intracellular C-terminus of the protein (De Jongh et al. 1991). Full-length _1S cRNA gave 10-fold smaller currents. Expression was also dependent on the presence of the _1b splice variant of the ₁ subunit, an isoform which had previously been thought to be localized to the brain (Hofmann et al. 1994) but which the authors showed to be a measurable transcript in rat muscle fibres.

The purpose of this study was to use the techniques devised by Ren & Hall (1997) to express the wild-type skeletal muscle L-type Ca²⁺ channel and all three HypoPP mutants in Xenopus oocytes in order to compare their effects on L-type channel gating in the same expression system. The mutations all affected the G(V) curve and the kinetics of activation and deactivation but did not significantly affect the voltage dependence of inactivation. The biophysical effects of the domain IV mutations R1239H and R1239G were markedly different from those of the domain II mutation R528H, implying that the location of the HypoPP mutations is an important determinant of how they perturb channel gating.

	METHODS

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Molecular biology

The rabbit _1S, rat brain _1b, 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, USA). The _1S and cDNAs were subcloned into the pGEMHE oocyte expression vector (gift of Dr Emily Liman, Harvard Medical School, Boston, MA, USA) at the HindIII and BamHI polylinker sites, respectively. The _1b cDNA was subcloned into pGEMHE between the SacII and HindIII sites. The ₂ cDNA was left in the pcDNA3 vector for oocyte expression. To obtain substantial L-type Ca²⁺ channel expression, it was necessary to truncate the C-terminus of the _1S cDNA at codon 1698, following Ren & Hall (1997). _1SC-pGEMHE was made from _1S-pGEMHE using the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA, USA). The mutagenic primer caused a loop-out deletion of base pairs 5320-5844 (amino acids 1698-1893) of the published rabbit _1S sequence, while the selection primer mutated the MfeI site at position 2788 of the pGEMHE sequence to a unique NdeI site. The three HypoPP point mutations, R528H, R1239H and R1239G, were introduced into _1SC-pGEMHE using the Transformer kit. In this case, the selection primer was designed to mutate the unique NdeI site in the vector back to an MfeI site.

For in vitro synthesis of RNA, the plasmids containing the Ca²⁺ channel subunits were linearized using the following restriction enzymes: Sse8387I for _1S, _1SC, _1SC-R528H, _1SC-R1239H and _1SC-R1239G (all in pGEMHE); NotI for _1b-pGEMHE; PvuII for ₂-pcDNA3; and NheI for -pGEMHE. Capped cRNA coding for all subunits was synthesized using the mMessage mMachine T7 kit (Ambion, Austin, TX, USA) and cleaned using the RNAid purification kit (Bio101, Vista, CA, USA).

Xenopus oocyte expression and electrophysiology

Stage V and VI oocytes were harvested from egg-bearing female Xenopus laevis frogs under anaesthesia with 3-aminobenzoic acid ethyl ester (1 mg ml^-1 in a cold water bath for 25 min; Sigma). After oocyte removal, the abdominal incision was re-sutured and the frog was allowed to recover for 1-3 h in a small volume of bottled spring water (Belmont Spring, Belmont, MA, USA). Frogs were maintained between oocyte harvests in 15-gallon tanks (< 10 frogs per tank) containing clean bottled spring water and were fed once per week with Frog Brittle (NASCO, Fort Atkinson, WI, USA). All experiments were performed in accordance with the guidelines of the Subcommittee on Research Animal Care of the Committee on Research of the Massachusetts General Hospital (protocol accession no. 99-4031). 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, Gathersburg, MD, USA) for 2·5 h in a room temperature shaker (set at 60 r.p.m.) 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 and 5 Hepes, with 50 µg ml^-1 gentamicin (Gibco BRL), pH 7·6.

Oocytes were injected with 50-100 nl of a 1:1:1:1 mixture of _1SC (or _1S), _1b, ₂ and cRNA using injection pipettes pulled from thin-wall capillary glass (no. 4878; World Precision Instruments, Sarasota, FL, USA) and were stored at 18°C for 5-7 days before use. For two-electrode voltage clamp recording, oocytes were placed in a bath solution containing (mM): 10 Ba(OH)₂, 96 NaOH and 10 Hepes, adjusted to pH 7·0 with methanesulfonic acid. Voltage-dependent Ba²⁺ currents were recorded using a Warner OC-725C amplifier (Warner Instrument Corp., Hamden, CT, USA) under the control of a custom-made stimulation/recording program written in AxoBASIC and running on an IBM-compatible computer. Current traces were leak subtracted on-line, filtered at 1 kHz and sampled at 2 kHz. Recording electrodes, fabricated from borosilicate capillary glass (1·65 mm outer diameter; VWR Scientific, West Chester, PA, USA) using a multistage puller (Sutter Instrument Co., Novato, CA, USA), were filled with 3 M KCl and had resistances in bath solution of 0·2-2 M. All recordings were made at room temperature (22-25°C). (-)-Bay K8644 (Bay K; Sigma) and nimodipine (gift of Dr Bruce Bean, Harvard Medical School) were stored at a stock concentration of 3 mM in DMSO and diluted in bath solution to a final concentration of 5 µM.

Data analysis

Current traces were digitally filtered off-line and analysed using custom-made software written in AxoBASIC. Curve fitting was accomplished in AxoBASIC or using SigmaPlot (Jandel Scientific, San Rafael, CA, USA). Data in the figures or reported in the text are presented as means ± S.E.M.

	RESULTS

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Expression of the skeletal muscle L-type Ca²⁺ channel in Xenopus oocytes

Figure 1A shows inward Ba²⁺ currents measured in response to a family of depolarizing voltage steps when the _1b, ₂ and subunits were co-expressed with the truncated _1S subunit (_1SC), with full-length _1S, or without an exogenous subunit. Figure 1B shows mean I(V) curves measured for these cases, along with the mean I(V) curve in uninjected oocytes. Expression of _1SC plus _1b, ₂ and gave rise to a slowly activating and inactivating voltage-dependent current that resembled the slow L-type Ca²⁺ current seen in native skeletal muscle. The current was sensitive to the dihydropyridine drugs nimodipine and (-)-Bay K8644 applied at a concentration of 5 µM (which reduced and boosted the current, respectively), a further indication of the presence of L-type channels (data not shown). Peak current, which occurred at +10 mV, was typically between 200 and 500 nA but reached 800-900 nA in several oocytes (mean, 469 ± 48 nA). Expression of the auxiliary subunits without the _1S subunit gave rise to a quickly activating and inactivating inward current, presumably representing Ba²⁺ flow through an endogenous Ca²⁺ channel whose functional expression was augmented by the exogenous auxiliary subunits. The endogenous current reached a peak of 185 ± 31 nA at +10 to +15 mV and was insensitive to 5 µM nimodipine or 5 µM Bay K (data not shown). Uninjected oocytes showed no appreciable inward Ba²⁺ current. Elimination of the _1b or ₂ subunit abolished the inward Ba²⁺ current completely, while elimination of the subunit did not abolish the current but reduced it significantly (data not shown). The current measured when the auxiliary subunits were expressed with full-length _1S was 8- to 10-fold smaller than the current conducted by _1SC and showed rapid activation and inactivation kinetics resembling those of the endogenous current. However, this current was consistently smaller than that observed with expression of the auxiliary subunits alone. This suggests that the exogenous _1S subunit, while passing little Ba²⁺ current, was present in the oocyte membrane and competed with the endogenous ₁ subunit for the auxiliary subunits.

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Figure 1. L-type channel expression in Xenopus laevis oocytes A, Ba2+ currents were evoked by step depolarizations to several test potentials (shown in the inset) from a holding potential of -90 mV when the three auxiliary Ca2+ channel subunits (1b, 2 and ) were expressed with 1SC (left panel), with 1S (upper right panel), or alone (lower right panel). Currents were recorded in 10 mM Ba2+ bath solution, leak subtracted on-line, and filtered at 1 kHz. At the onset and offset of the voltage step, 3 ms of each current trace was blanked in order to remove the residual capacitive transient. The dotted lines show the zero current level. B, the peak inward current was measured as a function of voltage for uninjected oocytes () and for oocytes expressing the auxiliary subunits alone (), with 1S (), or with 1SC ().

The HypoPP mutations reduce current density and alter the voltage dependence and kinetics of activation

In order to study the effects of the HypoPP mutations R528H, R1239H and R1239G on the gating properties of the skeletal muscle L-type Ca²⁺ channel, we introduced these mutations into the wild-type _1SC construct (_1SC-WT) and expressed _1SC-WT, _1SC-R528H, _1SC-R1239H and _1SC-R1239G with the _1b, ₂ and subunits in oocytes. Because the total current measured in each case represented a mixture of L-type and endogenous Ba²⁺ current, we used nimodipine subtraction to isolate the L-type current. Identical voltage families were applied before and after exposure of the oocyte to 5 µM nimodipine (a concentration sufficient for complete block of L-type current), and the traces recorded in nimodipine were subtracted from those recorded in the absence of the drug to give the nimodipine-sensitive current as a function of voltage. Figure 2 shows examples of the nimodipine-sensitive current recorded from oocytes expressing _1SC-WT, _1SC-R528H, _1SC-R1239H and _1SC-R1239G in response to a series of voltage pulses from the holding potential of -90 mV. The amplitude of the L-type current was consistently smaller for the mutant subunits than for the WT subunit, while the size of the endogenous current left after nimodipine block was roughly the same in each case (50-80 nA; not shown). The reduction of L-type current was more severe for the domain IV S4 mutations (R1239H and R1239G) than for the domain II S4 mutation (R528H).

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Figure 2. Nimodipine-sensitive L-type Ba2+ currents in oocytes expressing 1SC-WT, 1SC-R528H, 1SC-R1239H and 1SC-R1239G Ba2+ currents were evoked by a series of test pulses from the holding potential of -90 mV (see inset) before and after bath application of 5 µM nimodipine. The currents recorded in nimodipine were subtracted from the corresponding currents recorded before nimodipine application to give the nimodipine-sensitive current, shown in the figure. The currents were leak subtracted and filtered at 500 Hz, and 3-4 ms of each current record was blanked at the onset of the voltage step to remove the residual capacitive transient. The dotted lines represent the zero current level.

Figure 3A shows the mean I(V) curves for the nimodipine-subtracted current measured in oocytes expressing _1SC-WT, _1SC-R528H, _1SC-R1239H and _1SC-R1239G. The mean WT L-type current amplitude measured in these experiments (I_peak = -226 ± 19 nA) was significantly lower than that presented in Fig. 1, in which the mean L-type plus endogenous current amplitude was -469 ± 48 nA. This discrepancy arose from several very large (800 nA) L-type currents which were included in Fig. 1 but not used for nimodipine-subtraction experiments.

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Figure 3. Voltage-dependent activation of nimodipine-sensitive L-type Ba2+ currents conducted by 1SC-WT, 1SC-R528H, 1SC-R1239H and 1SC-R1239G The nimodipine-sensitive current was isolated as described in the legend of Fig. 2. A-C: WT, and continuous line; R528H, and dotted line; R1239H, and dashed line; R1239G, and dot-dot-dashed line. A, mean I(V) curves. The lines are cubic spline curves to guide the eye and are not fits of a model for the I(V) relationship. B, mean G(V) curves calculated from the data in A. Individual I(V) curves were fitted with the function: I(V) = {Gmax(V - Vrev)}/{1 + exp[-(V - V½)/k]}, whereVrev is the reversal potential for Ca2+ current, Gmax is the maximal conductance, V½ is the half-activation voltage and k is a steepness factor. Normalized G(V) curves were obtained by dividing the I(V) curves by Gmax(V - Vrev). WT: Vrev = 61·8 ± 2·9 mV, V½ = -0·2 ± 0·9 mV, k = 10·6 ± 0·1 mV. R528H: Vrev = 43·9 ± 1·9 mV, V½ = -5·7 ± 1·0 mV, k = 11·8 ± 0·2 mV. R1239H: Vrev = 47·4 ± 2·7 mV, V½ = +10·0 ± 0·9 mV, k = 8·8 ± 0·8 mV. R1239G: Vrev = 41·7 ± 3·8 mV, V½ = +6·5 ± 1·3 mV, k = 11·5 ± 0·7 mV. C, the voltage dependence of the rate of activation was measured by fitting the nimodipine-subtracted currents used in A and B with a function describing independent single-exponential activation and inactivation processes: I(t) = A{1 - exp[-(t - Td)/act]}exp[-(t - Td)/h], where A is the current amplitude, Td is the delay before the onset of the current, act is the time constant of activation and h is the time constant of inactivation. The figure shows act as a function of test voltage for WT, R528H, R1239H and R1239G.

On average, R528H reduced the amplitude of nimodipine-subtracted L-type current by 40 % compared with WT (I_peak was -138 ± 12 nA for R528H), while the domain IV mutations reduced the current by 60-70 % (I_peak was -84 ± 7 nA for R1239H and -73 ± 4 nA for R1239G). The peak of the I(V) curve occurred at +10 to +15 mV for the WT current but was left-shifted for the R528H current, occurring at 0 to +5 mV. The peak was slightly right-shifted for R1239H and was unchanged for R1239G.

In Fig. 3B, the I(V) curves have been transformed to normalized G(V) curves by fitting them with a Boltzmann distribution multiplied by a linear open-channel I(V) function (details in the legend of Fig. 3). R528H shifted the midpoint of the G(V) curve 5 mV to the left (V_½ = -5·7 ± 1·0 mV for R528H vs. -0·2 ± 0·9 mV for WT). R1239H had the opposite effect, causing an even greater shift to the right (V_½ = +10·0 ± 0·9 mV); R1239G also appeared to cause a rightward shift (V_½ = +6·5 ± 1·3 mV). In the fits, the reversal potential (V_rev) estimated for the WT currents was substantially more positive than the values estimated for the mutant currents (values given in the legend of Fig. 3). Since it is unlikely that the mutations affected the selectivity properties of the L-type Ca²⁺ channel, this was most likely caused by the smaller amplitude of the mutant currents, which made them susceptible to superimposed outward currents at the most depolarized test potentials. Even though the currents were nimodipine subtracted and our external solution was Cl^- free (see Methods), it is possible that small Ba²⁺-activated Cl^- currents contaminated some of the recordings. To assess the effect of the differences in V_rev on our G(V) curve fits, we re-fitted all of the mutant I(V) curves with V_rev fixed at the mean WT estimate of +61·9 mV and calculated a new set of G(V) curves. The leftward and rightward G(V) shifts caused by R528H and R1239H, respectively, were unchanged by this adjustment, making us confident that these were genuine effects of the mutations. The rightward shift caused by R1239G, however, was eliminated by adjusting V_rev to the WT value. Thus, it is possible that the shift shown in Fig. 3B for the R1239G currents is an artefact of our underestimate of V_rev for this mutant.

To measure the rate of L-type current activation, we fitted the activation time course with a function describing independent single-exponential activation and inactivation processes and measured the time constant of activation (_act) as a function of test voltage between -20 and +30 mV (details in the legend of Fig. 3). As shown in Fig. 3C, activation of the WT currents became faster with depolarization, accelerating by a factor of 3 in this voltage range. All three of the HypoPP mutations disrupted this voltage dependence. For the R528H currents, as for WT, activation became faster with increasing depolarization, although the voltage dependence of _act for the R528H currents was shallower than for WT. For R1239H and R1239G, _act could be measured reliably only above -10 mV due to the small size of the nimodipine-subtracted currents. For these mutants, the voltage dependence of the activation rate was markedly changed: _act became slower, rather than faster, at more depolarized potentials. The disruption was particularly severe for the R1239H currents, whose rate of activation slowed by a factor of 1·5 between -5 and +20 mV (_act was 22·3 ± 1·7 ms at -5 mV and 34·0 ± 3·1 ms at +20 mV).

The HypoPP mutations do not alter the voltage dependence of steady-state inactivation

Figure 4A shows currents recorded at +5 mV from an oocyte expressing _1SC-WT and an oocyte expressing _1SC-R528H after a series of 60 s conditioning pulses. The amount of inactivation produced by the conditioning pulse was quantified by measuring the current amplitude at 400 ms, a time point at which any current contribution from endogenous channels would have considerably inactivated (see Fig. 1A). Consequently, nimodipine subtraction was not used to measure steady-state inactivation. For both WT and R528H currents, inactivation was roughly 50 % complete at -50 mV and was 85 % complete at -10 mV. Figure 4B shows the mean voltage dependence of steady-state inactivation for WT and R528H L-type currents, measured as in Fig. 4A. The WT and R528H steady-state inactivation curves had very similar midpoints (V_½ = -47·1 ± 1·5 mV for R528H vs. -45·4 ± 1·8 mV for WT) and were of similar steepness (k = 15·1 ± 1·4 mV for R528H vs. 17·1 ± 0·7 mV for WT). In a few examples in which nimodipine subtraction was used to isolate WT and R528H L-type currents, the voltage dependence of steady-state inactivation was essentially the same (data not shown).

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Figure 4. The voltage dependence of steady-state inactivation for WT and R528H L-type Ba2+ current A, Ba2+ currents were measured at +5 mV in an oocyte expressing 1SC-WT (left panel) and an oocyte expressing 1SC-R528H (right panel) after 60 s conditioning pulses to a range of voltages (inset). Oocytes were held at -90 mV for 90 s between pulses. The currents were leak subtracted on-line and filtered at 500 Hz. The dashed lines show the zero current level. B, steady-state inactivation curves. Families of Ba2+ currents were generated using the voltage protocol shown in A. The current was measured at the end of each 400 ms test pulse to minimize contamination by the rapidly inactivating endogenous Ba2+ current. The current values were fitted to the Boltzmann function I = {(Imax - p)/[1 + exp((Vcond - V½)/k)]} + p, where V½ is the half-inactivation voltage, k is the steepness factor and p is the steady-state fraction of channels available at positive voltages. The values were then normalized by the fitted value of Imax and averaged to give the steady-state inactivation curves shown. The lines show the mean Boltzmann fits. WT ( and continuous line): V½ = -45·4 ± 1·8 mV, k = 17·1 ± 0·7 mV, p = 0·05 ± 0·03. R528H ( and dashed line): V½ = -47·1 ± 1·5 mV, k = 15·1 ± 1·4 mV, p = -0·007 ± 0·02.

The low signal-to-noise ratio observed in oocytes expressing _1SC-R1239H and _1SC-R1239G made it more difficult to measure the voltage dependence of steady-state inactivation for these mutants. We therefore compared the steady-state inactivation of WT, R1239H and R1239G L-type current in the presence of the dihydropyridine agonist (-)-Bay K8644 (Bay K; 5 µM), which boosted the current level and made the Ba²⁺ current less vulnerable to small leak changes during the long conditioning pulses. Figure 5A shows examples of Bay K-enhanced WT, R1239H and R1239G test currents after a series of 60 s conditioning pulses, and Fig. 5B shows the corresponding mean steady-state inactivation curves. The WT, R1239H and R1239G Boltzmann curves had similar midpoints (V½ = -38·1 ± 1·5 mV for R1239H and -45·3 ± 2·4 mV for R1239G, vs. -42·2 ± 2·5 mV for WT) and slope factors (k = 17·6 ± 0·9 mV for R1239H and 20·2 ± 1·7 mV for R1239G, vs. 15·2 ± 2·4 mV for WT). However, fitting the data with a Boltzmann function plus a constant demonstrated a larger fraction of channels available at depolarized potentials for R1239G (0·15 ± 0·02) compared with R1239H (0·04 ± 0·03) and WT (0·08 ± 0·02).

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Figure 5. The voltage dependence of steady-state inactivation of WT, R1239H and R1239G L-type current in the presence of the dihydropyridine agonist Bay K A, families of Ba2+ currents were recorded in the presence of 5 µM Bay K from oocytes expressing 1SC-WT (top panel), 1SC-R1239H (middle panel) and 1SC-R1239G (bottom panel). The currents were evoked by test pulses to +5 mV after a series of 60 s conditioning pulses from the holding potential of -90 mV; the conditioning voltages are indicated at the right of the top panel. Currents were leak subtracted on-line and filtered at 300 Hz. In each case, 3-4 ms of the trace was blanked at the start of the step to +5 mV to remove a residual capacitive transient. The dashed lines show the zero current level. B, the voltage dependence of steady-state inactivation in the presence of 5 µM Bay K. Families of Bay K-enhanced L-type currents were recorded as in A, and the current was measured at the end of each 300 ms test pulse. The measured current values were fitted to Boltzmann functions (lines in the figure) as described in the legend of Fig. 4. WT ( and continuous line): V½ = -42·2 ± 2·5 mV, k = 15·2 ± 2·4 mV, p = 0·08 ± 0·02. R1239H ( and dashed line): V½ = -38·1 ± 1·5 mV, k = 17·6 ± 0·9 mV, p = 0·04 ± 0·03. R1239G ( and dot-dot-dashed line): V½ = -45·3 ± 2·4 mV, k = 20·2 ± 1·7 mV, p = 0·15 ± 0·02.

The HypoPP mutations alter the activation and deactivation of Bay K-enhanced Ba²⁺ current

In order to confirm the effects of the HypoPP mutations on L-type current activation in the setting of a better signal-to-noise ratio and to record tail currents, we recorded WT and mutant I(V) curves in the presence of 5 µM Bay K. Figure 6 shows the voltage dependence of activation of Bay K-enhanced WT, R528H, R1239H and R1239G L-type current. As shown in the examples in Fig. 6A (compared with the nimodipine-subtracted currents of Figs 2 and 3), Bay K boosted the peak current 5- to 7-fold for both WT and mutant channels and greatly slowed the kinetics of tail currents observed upon repolarization to -90 mV. Figure 6B shows normalized G(V) curves measured in the presence of Bay K. The agonist caused a leftward shift and an increase in the steepness of the G(V) curves for all of the currents without changing the relative effects of the mutations (compare with Fig. 3B). Just as without Bay K, the midpoint of the R528H G(V) curve was shifted 5 mV to the left (V_½ = -8·9 ± 0·9 mV for R528H vs. -4·7 ± 1·3 mV for WT), while the R1239H and R1239G curves were shifted > 5 mV to the right (V_½ = +1·5 ± 1·0 mV for R1239H and +2·2 ± 0·7 mV for R1239G). In this case, the fitted V_rev values were only modestly different for WT and mutant currents (see Fig. 6 legend), and the relative positions of the WT and mutant G(V) curves were unchanged when the curves were re-fitted with V_rev fixed to the WT value (not shown).

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Figure 6. Voltage-dependent activation of L-type Ba2+ currents recorded in the presence of Bay K A, L-type currents were recorded from oocytes expressing 1SC-WT (upper left panel), 1SC-R528H (upper right panel), 1SC-R1239H (lower left panel) and 1SC-R1239G (lower right panel) in the presence of 5 µM Bay K. Each panel shows the currents evoked by pulses from the holding potential of -90 mV to test potentials of -60, -40, -20, 0 and +20 mV. The currents were leak subtracted on-line and filtered at 1 kHz. The vertical scale bars are different for the mutant and WT currents. B, normalized G(V) curves were constructed using fits of a Boltzmann distribution plus a linear open-channel I(V) function to the I(V) curves, as described in the legend of Fig. 3. The Vrev values determined from the fits were 41·9 ± 1·9 mV for WT, 34·2 ± 1·6 mV for R528H, 39·3 ± 2·5 mV for R1239H and 35·1 ± 1·9 mV for R1239G. The lines show the mean Boltzmann functions. WT ( and continuous line): V½ = -4·7 ± 1·3 mV, k = 8·3 ± 0·3 mV. R528H ( and dotted line): V½ = -8·9 ± 0·9 mV, k = 9·4 ± 0·1 mV. R1239H ( and dashed line): V½ = +1·5 ± 1·0 mV, k = 8·6 ± 0·1 mV. R1239G ( and dot-dot-dashed line): V½ = +2·2 ± 0·7 mV, k = 10·1 ± 0·2 mV.

The tendency of Bay K to slow the kinetics of deactivation made it possible to measure tail currents reliably, which allowed us to gain a more complete picture of activation gating in the WT and mutant channels. The left panel of Fig. 7A shows examples of Bay K-enhanced WT and R528H tail currents at two voltages, normalized by the extrapolated peak tail current amplitude (see Fig. 7 legend). The right panel shows examples of WT and R528H activation time courses, normalized by the maximum inward current during the test pulse. Tail currents were fitted with single-exponential functions, and activation time courses were fitted with functions describing superimposed single-exponential activation and inactivation processes, as in Fig. 3; the traces in Fig. 7A are labelled with their fitted _tail or _act values. Figure 7B shows the voltage dependence of _tail and _act for Bay K-enhanced WT and R528H currents. For both WT and R528H currents, _act was more steeply dependent on voltage in the presence of Bay K than in the absence of Bay K, primarily because of a slow component of activation introduced by Bay K at more hyperpolarized voltages (-20 to -5 mV). The lines are fits to the mean data points of a two-state model of activation and deactivation. In the model, the current relaxes after a voltage jump with a time constant given by 1/( + ), where and are the voltage-dependent forward and backward rate constants, respectively, connecting a single closed state to a single open state. and are saturating functions of voltage - i.e. at extreme positive voltages approaches _max and approaches zero, while at extreme negative voltages approaches _max and approaches zero (see Fig. 7 legend for details). The fits to the data in Fig. 7B suggest that the most prominent effects of the R528H mutation were to decrease _max and _max, giving slower limiting values of _tail at negative voltages and _act at positive voltages, and to shift the midpoint of the voltage dependence of (and hence of _act) to the left. These changes in the voltage dependence of _act are qualitatively similar to, though more severe than, the changes observed in the absence of Bay K (Fig. 3C).

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Figure 7. The kinetics of activation and deactivation of WT and R528H L-type Ba2+ currents enhanced by Bay K A, left panel, examples of tail currents recorded at -100 mV (top) and -60 mV (bottom) after a 200 ms conditioning pulse to +5 mV. Currents (means of five responses) were recorded from an oocyte expressing 1SC-WT (continuous lines) and an oocyte expressing 1SC-R528H (dotted lines) after bath application of 5 µM Bay K. Tail currents were fitted to a single-exponential function, and the fits were used to predict the peak tail current by extrapolating back to the end of the +5 mV conditioning pulse. tail values are shown for each trace. For comparison, the currents were normalized to the extrapolated peak tail current. The dashed lines show the zero current level. Right panel, examples of activation time courses recorded in 5 µM Bay K during test pulses to -10 mV (top) and +20 mV (bottom) from the holding potential of -90 mV. The activation time courses were fitted as described in the legend of Fig. 3; act is given in the figure for each trace. The WT currents (continuous lines) and R528H currents (dotted lines) were normalized to the peak inward current measured during the 300 ms test pulse. The dashed lines show the zero current level. B, comparison of the activation and deactivation kinetics of Bay K-enhanced WT and R528H currents over a range of voltages. The lines are fits to a two-state model of activation and deactivation. In the model, = 1/( + ), where and are the rate constants governing the opening and closing, respectively, of the channel. and are saturating functions of voltage: (V) = max/{1 + exp[-(V - V½,)/k]} and (V) = max/{1 + exp[(V - V½ ,)/k]}, where max and max are the saturating values of and at extreme positive and negative voltages, respectively, V½, and V½, are the voltages at which the rate constants are half-maximal, and k and k describe the steepness of their voltage dependence. WT ( and continuous line): max = 1·82 ms-1, max = 0·16 ms-1, V½, = +83·5 mV, V½, = -102·9 mV, k = 20·2 mV and k = 31·2 mV. R528H ( and dotted line): max = 0·05 ms-1, max = 0·05 ms-1, V½, = -8·4 mV, V½, = -69·1 mV, k = 21·6 mV and k = 18·0 mV.

Figure 8A compares normalized tail currents (left panel) and activation time courses (right panel) recorded from oocytes expressing WT, R1239H or R1239G channels. The values of _tail or _act obtained from fits to the time courses are indicated. Tail currents decayed faster for both mutants. For small depolarizations (-20 to 0 mV) the mutant channels activated more quickly than the WT channels, whereas at large depolarizations the reverse was observed. Figure 8B compares the voltage dependence of _tail and _act for WT, R1239H and R1239G channels over a wide voltage range; the lines are fits to the same two-state model used in Fig. 7. _max was greatly decreased for both mutations (most strikingly for R1239H), causing a marked change in the voltage dependence of _act. _max was barely affected (giving near-normal values of _tail at extreme negative voltages), but the midpoint of the voltage dependence of was strongly shifted to the right, causing to be closer to _max (and _tail to be closer to its limiting value) over much of the voltage range. The relative severity of the effects of R1239H and R1239G on _act followed the pattern observed in the absence of Bay K (Fig. 3C).

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Figure 8. The kinetics of activation and deactivation of Bay K-enhanced R1239H and R1239G L-type Ba2+ currents as a function of voltage A, examples of tail currents (left panel) and activation time courses (right panel) recorded from oocytes expressing 1SC-WT (continuous lines), 1SC-R1239H (dashed lines) and 1SC-R1239G (dot-dot-dashed lines). The voltage protocols are shown in the inset to each panel. The currents were fitted and normalized as described in the legend of Fig. 7. The horizontal dashed lines show the zero current level. B, comparison of the activation and deactivation kinetics of WT, R1239H and R1239G currents over a range of voltages. The lines are fits to a two-state model of activation and deactivation, as described in the legend of Fig. 7. WT ( and continuous line): see Fig. 7 legend for fit parameters. R1239H ( and dashed line): max = 0·02 ms-1, max = 0·15 ms-1, V½, = +3·3 mV, V½, = -48·0 mV, k = 9·0 mV and k = 29·2 mV. R1239G ( and dot-dot-dashed line): max = 0·09 ms-1, max = 0·11 ms-1, V½, = +20·2 mV, V½, = -40·8 mV, k = 23·2 mV and k = 18·3 mV.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The goal of this study was to measure the effects of the three known HypoPP mutations on the gating properties of the skeletal muscle L-type Ca²⁺ channel expressed in Xenopus oocytes. While the R528H mutation has been introduced into _1S and the cardiac isoform _1C and expressed in mouse L cells and HEK cells, respectively (Lapie et al. 1996; Lerche et al. 1996), ours is the first report comparing all three mutations in the same expression system. It is also the first instance in which the mutations have been introduced into the truncated form of _1S (_1SC), which makes up most of the _1S expressed in muscle. Injection of cRNAs encoding _1SC plus the _1b, ₂ and auxiliary subunits gave whole-oocyte Ba²⁺ currents of 200-900 nA (200-500 nA in most cases), 50-80 nA of which was carried by an endogenous oocyte Ca²⁺ channel combining with the auxiliary subunits. The HypoPP mutations R528H, R1239H and R1239G all reduced the amplitude of L-type current. In principle, this could reflect a reduction in the level of channel expression, a decrease in the unitary conductance of the channel, a reduction in maximal open probability, or an impairment in the ability of the mutant channels to compete with the endogenous Ca²⁺ channel for the auxiliary subunits. While we consider either a decrease in expression or a reduction in open probability to be quite plausible, a decrease in unitary conductance is not expected given the location of the mutations, and a difference in subunit binding seems unlikely given the similar endogenous current amplitudes observed after nimodipine block of the WT and mutant currents. The mutations also caused significant shifts in the G(V) curve (-5 mV for R528H, +5 to +10 mV for R1239H and perhaps for R1239G) and altered the voltage dependence of _act at positive test potentials. These changes in activation properties were the same whether or not Bay K was present in the bath. _tail, which was measured with Bay K present to slow the rate of deactivation, was slowed by R528H and accelerated at most voltages by R1239H and R1239G. None of the mutations significantly affected the voltage dependence of steady-state inactivation.

While the L-type currents produced by expression of the rabbit _1SC subunit in oocytes shared many properties with the L-type currents found in mature muscle fibres and cultured myotubes, these currents were different from the L-type currents in cultured myotubes in several ways. First, compared with the L-type current measured in WT human myotubes under similar ionic conditions (Jurkat-Rott et al. 1998; Morrill et al. 1998), the G(V) curve for WT _1SC channels expressed in oocytes was shifted 10 mV to the left; the steady-state inactivation curve was shifted 30 mV to the left, in spite of the fact that the rate of inactivation of L-type current appeared slower in oocytes at depolarized test potentials. Second, while the rate of activation of L-type current in WT human myotubes was weakly voltage dependent and showed a slight slowing with depolarization (Jurkat-Rott et al. 1998; Morrill et al. 1998), _act was strongly voltage dependent and became faster with depolarization in oocytes. Third, we observed a striking 5- to 7-fold enhancement of WT and mutant L-type currents in oocytes in the presence of 5 µM (-)-Bay K, significantly greater than the 30 % enhancement of the L-type current observed when 5 µM (±)-Bay K was applied to mouse myotubes (Strube et al. 1998) or the 3-fold enhancement observed when 5 µM (±)-Bay K was applied to neonatal mouse myotubes (which included the effect of a simultaneous switch from 10 mM Ca²⁺ to 110 mM Ba²⁺ external solution; Dirksen & Beam, 1995) - a difference too great to be easily explained by the use of racemic (±)-Bay K in these previous studies. Fourth, unlike the L-type currents measured in _1SC-R528H-injected oocytes, the L-type currents in heterozygous R528H myotubes showed no leftward shift in the G(V) curve and showed a 3-fold slowing of activation at depolarized potentials, which exceeds the modest slowing of _act measured in oocytes expressing _1S-R528H in the absence of Bay K (Fig. 3) and the 2-fold slowing of _act measured in the presence of Bay K (Fig. 7). The recordings of Jurkat-Rott et al. (1998) from human R528H myotubes and dysgenic myotubes expressing rabbit _1S-R528H did show a leftward shift in the G(V) curve comparable to that reported here but revealed no effect of the mutation on activation kinetics.

Requirements for L-type current expression

In agreement with Ren & Hall (1997), we found that the truncated _1SC subunit and the _1b variant of the ₁ subunit were crucial for obtaining significant L-type current. However, the current levels we observed (0·5 ± 0·05 µA in 10 mM Ba²⁺ after 5-7 days) were significantly smaller than those observed in the previous study (2·2 ± 0·2 µA in 40 mM Ba²⁺ after 3 days, and up to 6 µA after 7 days; Ren & Hall, 1997), a difference too great to be explained solely by the difference in permeant ion concentration (Hess et al. 1986) and perhaps reflecting instead the different oocyte expression vectors used in the two cases (pGEMHE in the present study vs. pAGA2 in the previous study). The current produced by expression of full-length _1S was 10-fold lower, as seen in the previous study. Although the currents observed in oocytes expressing _1S were somewhat similar to the endogenous currents seen in the absence of any exogenous ₁ subunit, the presence of some L-type current was verified by observing a response to 5 µM Bay K, which had no effect on the endogenous current (data not shown). Elimination of the auxiliary subunits _1b and ₂ prevented any detectable L-type current, while elimination of significantly reduced the current without abolishing it. This contrasts with the previous finding that elimination of ₂ gave a reduced (but non-negligible) current and elimination of had no effect on the current level (Ren & Hall, 1997).

Our results and those of Ren & Hall (1997) could be taken as evidence that only a subset of dihydropyridine receptors (DHPRs) in muscle - those containing the truncated _1SC subunit as well as the _1b subunit - are effective carriers of Ca²⁺ current. Since the truncated form makes up 90 % of the _1S protein expressed in muscle (De Jongh et al. 1991) and the _1b splice variant constitutes 2·5 % of the total ₁ subunit message (Ren & Hall, 1997), the current-carrying channels may represent as little as 2 % of the total number of muscle DHPRs. The idea that only a small percentage of DHPRs are functional channels was first proposed by Schwartz et al. (1985) based on the observation that there appeared to be many more high-affinity dihydropyridine binding sites than Ca²⁺ channels contributing to the macroscopic Ca²⁺ current in frog muscle fibres. The subsequent discovery that the full-length form of _1S accounts for less than 10 % of the total pool of DHPRs led to the hypothesis that the full-length isoform conducts Ca²⁺ current, while the truncated form only functions as a voltage sensor (De Jongh et al. 1991). Expression of truncated _1S in dysgenic myotubes, however, gave L-type Ca²⁺ currents that were identical to those produced when the full-length _1S cDNA was injected (Beam et al. 1992). In agreement with this, our results and those of Ren & Hall (1997) suggest that the truncated form of _1S, rather than the full-length form, might be the species specialized for conduction, specifically when it occurs in a complex with the rare _1b subunit.

There are significant problems with the proposal that Ca²⁺-conducting DHPRs make up a small minority of the total. For example, the calculations of Schwartz et al. (1985) depended on the assumption that skeletal muscle L-type Ca²⁺ channels have a maximal open probability near 1, far above the P_o,max values of 0·08-0·1 and 0·19 that have been measured for single skeletal muscle channels (Ma et al. 1991; Mejia-Alvarez et al. 1991; Dirksen & Beam, 1995). Moreover, the ability of full-length and truncated _1S to conduct Ca²⁺ and the role of the _1a and _1b subunits may differ between muscle cells and oocytes, and it is possible that the _1b subunit protein is more abundant than the _1b transcript in muscle, so our data and those of Ren & Hall (1997) do not rule out the possibility that most DHPRs are functional Ca²⁺ channels. Further biochemical and physiological studies in muscle cells will be needed to decide conclusively whether there is a mismatch between the number of voltage-sensing and Ca²⁺-conducting L-type channels in muscle and to determine how the two size forms of _1S and the two splice variants of the ₁ subunit might be specialized.

Divergent effects of the domain II and domain IV HypoPP mutations

The effects of the domain IV mutations R1239H and R1239G on L-type channel gating differed markedly from those of the domain II mutation R528H. While R528H caused a shift of the G(V) curve to the left, R1239H (and possibly R1239G) caused a shift of comparable magnitude in the opposite direction. Moreover, Bay K-enhanced tail currents were slowed by R528H but were accelerated by both R1239H and R1239G. All three mutations slowed the rate of activation at depolarized test potentials, although the effect was more pronounced for R1239H and R1239G than for R528H. These effects were analysed further by fitting a two-state model to the values of _tail and _act measured in the presence of Bay K. R528H decreased both _max and _max, the limiting forward and backward rate constants, respectively, and shifted the voltage dependence of the forward rate constant, , to the left. In contrast, both R1239H and R1239G decreased _max, left _max unchanged, and strongly shifted the voltage dependence of the backward rate constant, , to the right. Application of Bay K increased the amplitude of WT and mutant currents equally and did not change their relative effects on the voltage dependence and kinetics of activation, suggesting that none of the mutations interfered with the actions of the agonist on L-type channel gating.

Our results suggest that the domain in which these mutations occur is more important than the degree of conservation of the amino acid substitutions in predicting their physiological effects. R528H and R1239H are identical substitutions near the extracellular ends of domain II S4 and domain IV S4, respectively, yet these mutations cause very different changes in the opening and closing rates of the channel. Moreover, R1239H and R1239G have remarkably similar effects over a wide voltage range, in spite of the fact that R1239H replaces an arginine with an imidazole group (partially charged at pH 7·0), while R1239G replaces the same conserved arginine with a bare -carbon. The fact that the effects of these S4 substitutions are critically determined by the domains in which they occur is consistent with mounting evidence from Na⁺ channels that the S4 regions of these multidomain proteins can move separately and are specialized (Chen et al. 1996; Mitrovic et al. 1998; Cha et al. 1999). Our data differ, however, from those of a recent study showing differential effects of charge neutralizations in the four S4 segments of a chimeric cardiac-skeletal L-type Ca²⁺ channel expressed in dysgenic mouse myotubes (Garcia et al. 1997). In that study, mutation to glutamine of some of the inner charged residues in domains II and IV S4, including the analogue of R1239, had no effect on the G(V) curve or on _act, which led the authors to conclude that the voltage sensors of domains I and III are more important than the voltage sensors of domains II and IV in controlling the voltage dependence of activation. Our data also appear to differ from chimeric studies in the dysgenic myotube system which identified parts of domain I as the critical determinants of the activation rate of skeletal and cardiac L-type channels (Tanabe et al. 1991; Nakai et al. 1994). Our results suggest that in the skeletal muscle channel at least some parts of the S4 regions of domains II and IV are determinants of the voltage dependence and rate of activation.

Implications for HypoPP

Our data, which show very different biophysical defects caused by the domain II and IV mutations, suggest at first glance that a diversity of gating alterations can lead to the common cellular and clinical features of the disease. R528H, R1239H and R1239G channels do, however, share two physiological defects in common: (i) current density is reduced; and (ii) activation is slowed at strongly depolarized test potentials. These same defects were observed previously in heterozygous human myotubes carrying the R528H mutation (Morrill et al. 1998). Moreover, previous recordings from human myotubes carrying the R1239H mutation (Sipos et al. 1995), mouse L cells expressing the R528H mutation in the rabbit _1S subunit (Lapie et al. 1996), and HEK cells expressing R528H in the rabbit _1C subunit (Lerche et al. 1996) have all shown a reduction in current density for the mutant channels. It is tempting to hypothesize that these two alterations - both of which would tend to reduce the net influx of Ca²⁺ through these channels during a series of muscle action potentials - are the ones that are most important for predisposing the muscle fibre to depolarization and paralysis. In this respect, it is interesting to note that the R1239H mutation, which causes the most severe changes in the voltage dependence of _act at depolarized voltages, is associated clinically with more complete penetrance in women, a lower age of onset, and lower potassium levels during paralytic attacks than the R528H mutation (Elbaz et al. 1995; Fouad, et al. 1997).

How might a reduction in Ca²⁺ influx through L-type Ca²⁺ channels produce excessive muscle depolarization and paralysis in the setting of a reduced extracellular potassium concentration? One possibility is that reduced Ca²⁺ entry during muscle activity affects the membrane potential in real time, perhaps through coupling to a Ca²⁺-sensitive channel with direct control over the resting potential (e.g. a Ca²⁺-activated potassium channel). Alternatively, a chronic reduction in Ca²⁺ influx through L-type channels during muscle development might alter the expression, distribution, or regulation of channels affecting the membrane potential, thereby indirectly predisposing the fibre to depolarization. The latter hypothesis is lent credence by recent evidence that the skeletal muscle ATP-sensitive potassium channel - the most abundant potassium channel in muscle fibres - exhibits abnormal sub-conductance states and an abnormal response to ADP in fibres biopsied from HypoPP patients heterozygous for the R528H mutation (Tricarico et al. 1999). Regardless of how altered Ca²⁺ influx might destabilize the membrane potential, a reduction of extracellular K⁺ could further predispose the cell to depolarization if there were potassium channels in the membrane that depended on a normal extracellular potassium concentration to remain open, as has been observed for neuronal potassium channels (Pardo et al. 1992).

It also remains possible that the most important effects of the HypoPP mutations have nothing to do with Ca²⁺ influx through the channel but instead change the excitation-contraction coupling function of the channel. While fura-2 calcium imaging experiments in human WT and R528H myotubes have revealed no difference in the time course or magnitude of depolarization-evoked Ca²⁺ release from the sarcoplasmic reticulum (Jurkat-Rott et al. 1998), no attempt has yet been made to measure gating currents from DHPRs carrying the HypoPP mutations. While the L-type channel density in human myotubes and in oocytes may be too low to allow the resolution of gating currents in these systems, vaseline-gap studies in biopsied mature human muscle fibres from patients expressing the three mutations might allow gating currents to be measured, perhaps in conjunction with Ca²⁺ transients.

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