JP-45/JSRP1 Variants Affect Skeletal Muscle Excitation–Contraction Coupling by Decreasing the Sensitivity of the Dihydropyridine Receptor

ABSTRACT

JP-45 (also JP45; encoded by JSRP1) is an integral protein constituent of the skeletal muscle sarcoplasmic reticulum junctional face membrane interacting with Ca_v1.1 (the α.1 subunit of the voltage-sensing dihydropyridine receptor, DHPR) and the luminal calcium-binding protein calsequestrin. Two JSRP1 variants have been found in the human population: c.323C>T (p.P108L) in exon 5 and c.449G>C (p.G150A) in exon 6, but nothing is known concerning the incidence of these polymorphisms in the general population or in patients with neuromuscular diseases nor the impact of the polymorphisms on excitation–contraction (EC) coupling. In the present report, we investigated the frequencies of these two JSRP1 polymorphisms in the Swiss malignant hyperthermia population and studied the functional impact of the variants on EC coupling. Our results show that the polymorphisms are equally distributed among malignant hyperthermia negative, malignant hyperthermia equivocal, and malignant hyperthermia susceptible individuals. Interestingly, however, the presence of either one of these JP-45 variants decreased the sensitivity of the DHPR to activation. The presence of a JSRP1 variant may explain the variable phenotype seen in patients with malignant hyperthermia carrying the same mutation and, more importantly, may counteract the hypersensitivity of EC coupling caused by mutations in the RYR1 gene.

Introduction

Skeletal muscle excitation-contraction (EC) coupling is a physiological process whereby an electrical signal (depolarization of the plasma membrane) is converted into a chemical signal, that is, a calcium gradient, by the opening of ryanodine receptor (RyR1) Ca²⁺ release channels located on the sarcoplasmic reticulum (SR) terminal cisternae. The Ca²⁺ then binds to the contractile proteins to initiate muscle contraction; muscle relaxation occurs when the myoplasmic Ca²⁺ is pumped back into the SR by the activity of the SERCA CaATPases [Fleischer and Inui, 1989; Rios and Pizarro, 1991; Franzini-Armstrong and Jorgensen, 1994]. The two core components of the EC coupling machinery are the voltage sensing dihydropyridine receptor (DHPR) Ca²⁺ channel located on the transverse tubules and the RyR1. Upon sensing a change in voltage, the DHPR undergoes a conformational change whereby it directly interacts with and activates the RyR1, causing Ca²⁺ release from the SR [Nakai et al., 1996; Sutko and Airey, 1996]. The activity of the DHPR can be measured electrophysiologically and is accompanied by a measurable intermembrane current, which is proportional to the movement of charged ions across the transverse tubular membrane [Adams et al., 1990; Schneider and Chandler, 1973].

Several congenital neuromuscular disorders are linked to dysregulation of calcium homeostasis among which the subclinical myopathy malignant hyperthermia (MH; MIM# 145600), the congenital core myopathy central core disease (CCD; MIM# 117000), and some forms of multiminicore disease (MmD; MIM# 255320), centronuclear myopathy (CNM; MIM# 160150), and congenital fiber type disproportion [Clarke et al., 2010; Jungbluth et al., 2011; Treves et al., 2008; Wilmhurst et al., 2011]. More than 200 mutations in the RYR1 gene have been identified in patients belonging to these disease categories [Robinson et al., 2006; Jungbluth et al., 2011; Duarte et al., 2011], analysis of other genes encoding proteins involved in EC coupling have so far identified only mutations in CACNA1S, the gene encoding the α1 subunit (Ca_v1.1) of the skeletal muscle DHPR to be causatively linked to the pharmacogenetic disorder MH [Monnier et al., 1997; Pirone et al., 2010; Stewart et al., 2001; Toppin et al., 2010]. However, mutations in these genes only accounts for approximately 70–80% of the human cases and the search for mutations in other genes encoding proteins involved in EC coupling has so far yielded few if any true positive candidates.

MH is a subtle pharmacogenetic disorder triggered by halogenated anesthetics and the muscle relaxant succinylcholine in genetically predisposed individuals [Glahn et al., 2010; Robinson et al., 2006; Treves et al., 2008]. In their everyday life, such individuals are unaware of their condition though a small number of them may suffer from heat intolerance, muscle cramping after intense exercise, and rhabdomyolysis; however, once such individuals come in contact with a trigger agent they undergo a rapid hypermetabolic reaction which is often fatal if left untreated. Thus, identifying potentially susceptible patients and their relatives prior to contact with a anesthesia is of utmost importance and the aim of clinical MH-related research for noninvasive testing. In the past few years, novel proteins of the SR have been identified and in many cases their ablation has been shown to affect Ca²⁺ homeostasis and/or muscle performance. In previous studies, we identified JP-45, an integral membrane protein of skeletal muscle SR junctional face membrane which interacts via its amino terminal with Ca_v1.1 and via its carboxy terminal, with the luminal calcium binding protein calsequestrin [Anderson et al., 2003, 2006]. Three months old JP-45 mice showed decreased muscle strength, a decrease of the functional expression of the Ca_v1.1 and a decrease of depolarization-induced Ca²⁺ release [Delbono et al., 2007]. These changes however, were no longer evident in old (>12 month old) JP-45 KO mice [Delbono et al., 2012]. An initial study aimed at screening the JSRP1 gene (MIM# 608743), the gene encoding JP-45, showed the existence of two JP-45 variants (dbSNP and Exome Variant Server NHLBI Exome Sequencing Project databases): c.323C>T (exon 5) resulting in the p.P108L substitution (dbSNP database accession NM_144616.3 and NP_653217.1; rs74521370) and c.449G>C resulting in the p.G150A substitution (exon 6) (dbSNP data accession NM 144616.3 and NP653217.1; rs80043033) [Althobiti et al. 2009]; because of the prevalence of the latter polymorphism in MH susceptible individuals, it was hypothesized that such variants may serve as “function modifiers,” but no information as to how this could be accomplished was provided.

In the present study, we investigated (1) the frequency of the two JP-45 variants in the Swiss MH population and (2) the effect of the polymorphisms on EC coupling. Our results show that though the distribution does not vary significantly between MH negative (MHN), MH equivocal (MHE), and MH susceptible (MHS) individuals, the presence of either the p.P108L or the p.G150A variant affects the functional properties of the voltage sensor, without directly affecting the functional properties of the RyR1. Thus the presence of JSRP1 variants may downregulate the activity of the EC coupling machinery and may account for some of the phenotypic differences seen in individuals carrying the same RYR1 mutation.

Materials and Methods

Patient Selection and Genomic DNA Isolation

Patients referred to the Swiss MH investigation unit (Department of Anesthesia, University Hospital Basel, Switzerland) for MH screening using an open muscle biopsy were included in this study. All patients gave their written informed consent for storing their muscle tissue, establishing primary muscle cell cultures and using DNA obtained from a blood sample for research projects specifically focusing on MH. This procedure was approved by the Ethics Committee of the University Hospital Basel. The patients were classified into MHN, MHE, and MHS by the vitro contracture test (IVCT) on isolated muscle strips of the open muscle biopsy obtained from the vastus medialis or lateralis muscles under regional anesthesia. The IVCT was performed according to the protocol of the European MH Group [EMHG, 1984]. Individuals were diagnosed as MHS if a contracture of 0.2 g or greater occurred at 2 mM caffeine or less and 2% halothane or less. An MHN diagnosis was established if a contracture of 0.2 g was not reached by 2 mM caffeine and 2% halothane. An MHE diagnosis was made if a contracture of 0.2 g or greater occurred only at 2 mM caffeine or less (MHEc) or 2% halothane or less (MHEh) but not with both testing substances. Patients were also screened for the presence of RYR1 mutations included in the EMHG guidelines as previously described [Girard et al. 2001]. MH testing was approved by the ethical board of the Basel University Hospital. The presence of the JSRP1 polymorphic variants was assayed on genomic DNA isolated either from whole blood or untreated muscle tissue using QIAamp DNA mini kit (Qiagen AG, Hombrechtikon, Switzerland) following the manufacturers recommendations.

Identification of JSRP1 Variants

The following primers and conditions were used to amplify JP-45 from genomic DNA: exon F 5′- acagagccagtgaccacagc-3′ and R 5′-GAGGAATAGGCGCACAGGT-3′; exon 6 F 5′-GAGGGATGGACAGTGTGGAC-3′ and R 5′-CTTTCCTTGGGGATGAAGGT-3′. Amplification conditions were 1 cycle 95°C 4 min followed by 35 cycles annealing (65°C c.323C>T and 63°C for c.449G>C, 45 sec), extension (72°C, 40 sec), denaturation (95°C, 30 sec), followed by a 5 min extension cycle at 72°C. The PCR-amplified DNA fragments were purified using Qiagen PCR purification columns, digested with MspA1l (c.323C>T in exon 5) or AciI (c.449G>C in exon 6) run on a 15% acrylamide gel and visualized by ethidium bromide staining. The DNA mutation numbering system is based on the cDNA reference sequence, with +1 as the A of the initiator ATG codon.

Human Myotube Cultures and Intracellular Ca²⁺ Measurements

Primary skeletal muscle cultures were established from fragments of muscle biopsies obtained from patients undergoing diagnostic testing, as previously described [Ducreux et al., 2004]. Cells were cultured on 0.17 mm thick glass coverslips in growth medium and induced to differentiate into myotubes by culturing them in DMEM plus 4.5 mg/ml glucose, 0.5% BSA, 10 ng/ml EGF, 0.15 mg/ml creatine, 5 ng/ml insulin, 200 mM glutamine, 600 ng/ml penicillin G and streptomycin, and 7 mM HEPES, pH 7.4 for 7–10 days.

For cytoplasmic calcium measurements, coverslip grown myotubes were either loaded with the ratiometric fluorescent Ca²⁺ indicator fura-2-AM or Fluo-4 AM (final concentration 5 µM) in differentiation medium for 30 min at 37°C, after which the coverslips were mounted onto a 37°C thermostatically controlled chamber which was continuously perfused with Krebs–Ringer medium. Online measurements were recorded using a fluorescent Axiovert S100 TV inverted microscope (Carl Zeiss GmbH, Jena, Germany) equipped with a 20x water-immersion FLUAR objective (0.17 NA), filters (BP 340/380, FT 425, BP 500/530) and attached to a Cascade 125+ CCD camera or with a Nikon TE2000 TIRF microscope equipped with a dry 20x Plan Fluor objective (0.17 N.A.), filters (ex 472/3; em 488, BP 525/30), and an electron multiplier Hamamatsu CCD C9100-13 camera as previously described [Treves et al., 2009]. Changes in fluorescence were analyzed using Metamorph imaging system and the average pixel value for each cell was measured as previously described [Ducreux et al., 2004; Treves et al., 2009]. Individual cells were stimulated by means of a 12- or 8-way 100-mm diameter quartz micromanifold computer controlled microperfuser (ALA Scientific instruments, Westbury, NY), as previously described [Ducreux et al., 2004].

Electroporation of Muscles from JP-45 KO Mice with the cDNA Carrying the Variants in Exon 5 or Exon 6

JP-45KO mice were used for plasmid electroporation according to DiFranco et al. (2006). Briefly, flexor digitorum brevis (FDB) muscles were injected with 5 µl of 2mg/ml hyaluronidase and injected 1 hr later with 20 µg DsRed-conjugated JP-45 mutants or wild-type cDNAs. Ten minutes later, two sterile, gold-plated acupuncture needles were placed under the skin on adjacent sides of the muscle. Twenty 100 V/cm, 20-ms square-wave pulses of 1-Hz frequency were applied to the muscle for one second each using a Grass stimulator (Grass S48; W. Warwick, RI).

Charge Movement and Calcium Current Recordings

FDB muscle fibers were transferred to a small flow-through Lucite chamber positioned on a microscope stage. Fibers were continuously perfused with the external solution (see below) using a push–pull syringe pump. Only fibers exhibiting clean surface and lack of evidence of contracture were used for electrophysiological recordings. Muscle fibers were voltage-clamped using an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA) in the whole-cell configuration of the patch-clamp technique [Hamill et al., 1981; Delbono et al., 2007]. Patch pipettes were pulled from borosilicate glass (Boralex, WPI, Sarasota, FL) using a Flaming Brown micropipette puller (P97, Sutter Instrument Co., Novato, CA) and then fire-polished to obtain electrode resistance ranging from 450 to 650 kΩ. In cell-attached the seal resistance was in the range of 1–4.5 GΩ (n = 50) and in the whole-cell configuration the values were between ∼100 MΩ. Only experiments with resistance more than 100 MΩ were included in the analysis. The pipette was filled with the following solution (mM): 140 Cs-aspartate; 2 Mg-aspartate₂, 0.2 or 10 Cs₂EGTA (ethylene glycol-bis(α-aminoethyl ether)-N,N,N′N′-tetraacetic acid), 10 HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), pH was adjusted to 7.4 with CsOH [Adams et al., 1990]. The external solution used for Ca²⁺ current recording contained (mM): 150 TEA (tetraethylammonium hydroxide)-CH₃SO₃, 2 MgCl₂, 2 CaCl₂, 10 Na-HEPES, and 0.001 tetrodotoxin [Delbono, 1992]. Solution pH was adjusted to 7.4 with CsOH. Both, the pipette and the bath solution, were selected based on the ease of membrane seal formation and cell stability over time. For charge movement recording, Ca²⁺ current was blocked with the external solution containing 0.5 mM Cd²⁺ and 0.3 mM La³⁺ [Adams et al., 1990].

Whole-cell currents were acquired and filtered at 5 kHz with pCLAMP 10 software (Molecular Devices). A Digidata 1440A interface (Molecular Devices) was used for A–D conversion. Membrane current during a voltage pulse, P, was initially corrected by analog subtraction of linear components. The remaining linear components were digitally subtracted online using hyperpolarizing control pulses of one-quarter test pulse amplitude (−P/4 procedure) as previously described for mouse muscle fibers [Delbono, 1992]. Four control pulses were applied before the test pulse. Charge movements were evoked by 25 ms depolarizing pulses from the holding potential (−80 mV) to command potentials ranging from −70 to 60 mV with 10mV interval. Intramembrane charge movement was calculated as the integral of the current in response to depolarizing pulses (charge on, Q_on) and is expressed per membrane capacitance (coulombs per farad). The complete blockade of the inward Ca²⁺ current was verified by the Q_on − Q_off linear relationship. For analysis of the relationship between charge movement and membrane voltage, data points were fitted to a Boltzmann equation of the form Q = Q_max⁄[1 + exp(V_1⁄2)V_m)⁄K], where Qmax is the maximal charge; V_1⁄2 is the charge half-activation potential; V_m is the membrane potential; and k is the steepness of the curve. For calcium currents, normalized data points to the maximum current amplitude were fitted to the following equation:

• 

where G_max is the maximum conductance, V is the membrane potential, V_r is the reversal potential, V_1/2 is the half-activation potential, z is the effective valence, F is the Faraday constant, R is the gas constant, and T is the absolute temperature.

Sarcoplasmic Reticulum Ca²⁺ Release

Dissociated FDB fibers were loaded with Oregon Greeen Bapta-5N (OGB-5N) via the patch pipette as described [Jimenez-Moreno et al., 2008]. The dye was allowed to diffuse for 20–30 min before fiber stimulation and after attaining the whole-cell voltage-clamp configuration. Intracellular OGB-5N transients were recorded using a Bio-Rad Radiance 2100 laser scanning confocal microscope (Zeiss, Oberkochen, Germany). Confocal microscopy allowed us to improve the signal/noise ratio under experimental conditions in which myoplasmic Ca²⁺ concentration was strongly buffered by 20 mM EGTA. This experimental manipulation also ensured a more accurate estimation of the Ca²⁺ release flux. Fibers were imaged through a C-Apochromat 40× water-immersion objective (NA 1.2, Zeiss) or a 20× Fluar (NA 0.75) using an argon laser at 488-nm excitation wavelength. The fluorescence emission was measured at 528 ± 25-nm wavelength. For most experiments, the laser was attenuated to 6–12% with a neutral density filter. Fibers were imaged in line-scan (x–t) mode. The fiber was always oriented parallel to the x scan direction. Linescan images were acquired with 256 pixels (0.236 µm/pixel) in the x-direction and 512 pixels (0.833 ms/pixel) in the t-direction. For image acquisition, we used LaserSharp 2000 software (Bio-Rad, Zeiss), and for the analysis of the image intensity profile, Image J software (NIH, Bethesda, MD). For analysis of the relationship between fluorescence and membrane voltage, data points were fitted to a Boltzmann equation of the form ΔF/F = ΔF/F_max⁄[1 + exp(V_1⁄2)V_m)⁄K, where ΔF/F_max is the maximal normalized fluorescence; V_1⁄2 is the ΔF/F half-activation potential; V_m is the membrane potential; and K is the steepness of the curve.

Statistical Analysis

Statistical analysis was performed using the Student's t-test for paired samples; means were considered statistically significant when the P value was <0.05. The Origin computer program (Microcal Software, Inc., Northampton, MA) was used for statistical analysis.

Results

Figure 1 shows the human and mouse JP-45 amino acid sequences and the location of the JSRP1 polymorphic variants. We first assessed the frequency of the JP-45 polymorphism in exon 5 and exon 6 in the Swiss MH population, by screening the genomic DNA of patients that had been tested genetically and phenotypically and classified as either MHN, MHEh (with abnormal response to halothane) with mutation in RYR1, MHEh with no identified mutation in RYR1, or MHS with a causative RYR1 mutation. Figure 2 (left panels) shows a schematic representation of the restriction sites generated by the presence of the c.323C>T substitution (top) and of the c.449G>C 6 (bottom). The panels on the right show PCR-amplified DNA from individuals with and without the polymorphisms, after digestion with MspA1l (c.323C>T, exon 5) and Acil (c.449G>C exon 6) while the frequency distribution of the polymorphisms among the different individuals is shown in Table 1. As indicated (1) the frequency of the two SNP is not different between individuals belonging to different groups and (2) the SNP in exon 6 is more frequent compared to that in exon 5. We also identified one MHN individual carrying both polymorphisms.

Table 1. Frequency of JP-45 Polymorphisms in the Swiss MH Population

	MHN	MHEh Mutation (−)	MHEh Mutation (+)	MHS
^a NCBI reference sequence NM_144616.3; DNA mutation numbering system is based on cDNA sequences. The frequency of the two SNP is not different between MHN and MHS groups. Carriers were all heterozygous for JP-45 polymorphisms.
Exon 5 c.323C>Ta (p.P108L)	2/63 (3%)	1/13 (8%)	0/7 (0%)	2/57 (3 %)
Exon 6 c.449G>C^§ (p.G150A)	24/63 (38%)	7/13 (54%)	3/7 (43%)	20/57 (35%)

Figure 1. Comparison of the amino acid sequences of human and mouse JP-45 and location of the polymorphic variants.

Figure 2. Left: Schematic representation of DNA fragments from wild type and polymorphic variants of JP-45 showing size of fragments and location of restriction sites. Right: Representative polyacrylamide gel from a wild type (−) and carrier of JP-45 variants after PCR amplification and restriction enzyme digestion. PCR conditions and primers were as described in the section “Materials and Methods.”

To verify if the JSRP1 variants influence the resting [Ca²⁺] and sensitivity of the ryanodine receptor to KCl-induced Ca²⁺ release, we loaded myotubes from MHN/MHE individuals with no RYR1 mutation, carrying either the p.P108L polymorphism (2 individuals), the p.G150A polymorphism (three MHN and three MHE individuals), with either fura-2 or fluo-4 and studied general aspects of calcium homeostasis. Figure 3A shows that the resting [Ca²⁺]_i of individuals carrying the p.G150A polymorphism were slightly lower than those carrying either no polymorphism or the p.P108L. It is difficult to interpret if such small changes (<10% of the fura-2 fluorescent ratio value) though statistically significant, could be the consequence of altered EC coupling, in particular, due to fine alterations of Ca_v1.1 function. We next assessed the response of these myotubes to different concentrations of KCl. As shown in Figure 3B, the sensitivity to KCl was very similar in myotubes carrying the p.P108L and p.G150A polymorphism, with an EC₅₀ of 36.5 ± 2.5 and 30.5 ± 1.0, respectively but this was shifted to higher KCl concentrations compared to myotubes carrying no polymorphisms (EC₅₀ 11.3 ± 6.8); however, the peak calcium release induced by KCl or via direct pharmacological activation of the RyR1 with 600 µM 4-chloro-m-cresol were not different (Fig. 3C).

Figure 3. Calcium regulation in myotubes from individuals carrying wild type or polymorphic variants of JP-45. A: Resting fura-2 fluorescent ratio. The resting [Ca²⁺]_i concentration was slightly but significantly reduced in cells from individuals carrying the p.G150A polymorphism. B: KCl-induced Ca²⁺ release curves in myotubes from control individuals (pooled from three MHN individuals; *_*), from individuals carrying the p.P108L JP-45 variant (pooled from two MHN individuals - • - • - • -), and from individuals carrying the p.G150A JP-45 variant (pooled values from three MHN and three MHEh with no RYR1 mutation · - · ▪ · - · ▪ · - · ▪ · - · ▪ · - ·). Each point represents the mean (±S.E.M.) percent increase in fluo-4 fluorescence obtained at the indicated KCl concentration calculated with respect to the value obtained by stimulating myotubes with 100 mM KCl (n = 5–12 averaged values per point). C: Peak increase in fura-2 fluorescence ratio induced by 60 mM KCl (light gray boxes) and 600 µM 4-chloro-m-cresol (dark boxes); results are the mean (±SEM) of 9–12 measurements.

Because JP-45 interacts with Ca_v1.1 we investigated in greater detail if the fine functions of the DHPR voltage sensor were affected by the polymorphisms. To this end, we reconstituted FDB muscles from JP-45 KO mice with constructs carrying wild type JP-45 or constructs in which each of the single variants had been inserted, and studied the charge movement, Ca²⁺ currents, and intracellular calcium. Figure 4 (panels A and B) shows the electrophysiological properties of single FDB fibers carrying the different variants. Fibers reconstituted with the cDNA carrying the p.P108L or p.G150A variants showed ∼18 mV or more than 20mV displacement of the charge movement-membrane voltage curve toward more positive potentials compared to wild type JP-45 (Table 2). These changes in charge movement were accompanied by significant changes in the voltage-dependence of the calcium current (Fig. 4B). Half-activation potential for p.P108L (22 ± 2.8 mV) and p.G150A (23 ± 3.1 mV) was found at more depolarized potentials than wild type JP-45 (2.2 ± 0.41). As the shift in charge movement and calcium current activation for the p.G150A and p.P108L variants was significantly shifted toward more positive potentials compared to wild type JP-45, we examined the voltage-dependence of SR Ca²⁺ release. Consistently, SR Ca²⁺ release shows a ∼18 mV shift toward more depolarized potentials induced by both JP-45 variants compared to JP-45 wild type (Fig. 4C and Table 2).

Table 2. Best Fitting Parameters Describing the Voltage-Dependence of Charge Movement and Changes in the [Ca²⁺]_i in FDB Fibers

	Charge movement
	Maximal Q (nC/µF)	V1/2 (mV)	K
Q, charge movement; F, Oregon Green Bapta-5N fluorescence; V1/2, half-activation potential; K, curve steepness. ^a P < 0.01 for p.G150A or p.P108L versus wild type (WT). Charge movement, n = 8–14 fibers from 4–5 mice per group; calcium current, n = 7–13 fibers per group from four mice per group; and intracellular calcium, n = 8–11 fibers from 3–4 mice per group.
WT JP-45	26 ± 2.9	−8.6 ± 1.1	18 ± 1.2
p.P108L	24 ± 2.6	9.7 ± 0.9a	16 ± 1.3
p.G150A	26 ± 3.1	18 ± 2.1a	20 ± 1.6
	Intracellular calcium
	Maximal ΔF/F	FV1/2 (mV)	K
WT JP-45	0.61 ± 0.07	−2.3 ± 0.33	10 ± 2.1
p.P108L	0.58 ± 0.03	14 ± 3.1a	8.8 ± 2.6
p.G150A	0.55 ± 0.06	16 ± 2.8a	9.3 ± 1.9

Figure 4. Charge movement, calcium current, and SR Ca²⁺ release in wild-type JP-45 and JP-45 variants. A: Normalized charge movement to maximal charge (Q_max)–V_m relationship. Data points were fitted to a Boltzmann equation (Methods) and best fitting parameters are included in Table 2 (n = 8–14 fibers from 4–5 mice per group). B: Normalized calcium current (I_ca) − V_m. Data points were normalized to peak current as described (see section “Materials and Methods”) (n = 7–13 fibers per group from four mice per group). C: Normalized Oregon Green Bapta (OGB)–5N–V_m relationship (n = 8–11 fibers from 3–4 mice per group).

Discussion

In the present paper, we investigated the impact of two naturally occurring variants of JSRP1 on EC coupling and report that their presence downregulates the activity of the DHPR leading to a shift in its sensitivity to activation; thus the presence of polymorphic variants of JSRP1 and potentially in other genes encoding SR proteins may fine tune skeletal muscle EC coupling and explain the variable phenotypes observed in MHS patients from different genetic backgrounds, with the same RYR1 mutation.

While analyzing the MHS population, Althobiti et al. (2009) identified two genetic variants of the JSRP1, the gene encoding the SR junctional face membrane protein JP-45, but concluded that it was unlikely that either polymorphism on their own could be causative of malignant hyperthermia and rather such variants may act as modifiers in MH susceptibility. The results of the present study support and extend these findings; analyzing DNA samples collected from 140 individuals of the Swiss population undergoing genetic or functional testing for MH susceptibility, we found that approximately 50% of the population (MHN, MHE, and MHS) carried the p.G150A polymorphism, and it is equally distributed among the different MH subgroups. On the other hand the p.P108L polymorphism is much less frequent, being present in only approximately 5% of the total population with no significant difference in its distribution among the different MH subgroups. Nevertheless, the absence of a direct impact of genetic variants on the predisposition to MH as determined by the in vitro contracture test (the gold standard used to determine MH susceptibility) does not exclude an indirect modulatory effect of JP-45 on the fine tuning of EC coupling.

In order to study the functional effects of the JP-45 polymorphisms we used two complementary approaches: (1) we studied calcium homeostasis in myotubes from MHN/MHE individuals negative for RYR1 mutations and endogenously expressing the variants and (2) reconstituted skeletal muscles from JP-45 KO mice [Delbono et al., 2007] with either wild type cDNA or the cDNA carrying either variant of JP-45 and studied the electrophysiological characteristics of the DHPR from isolated FDB fibres. Both approaches gave similar results in that the sensitivity of the voltage sensor was shifted to higher depolarizing voltages/KCl concentrations in cells carrying either variant. This result is not totally unexpected as: (1) JP-45 binds directly to the Ca_v1.1 through its NH₂-terminal domain and to calsequestrin via its COOH-terminus [Anderson et al., 2006]; (2) overexpression of JP-45 in C₂C₁₂ myotubes leads to a reduction of Ca²⁺ permeability per voltage-sensor charge [Gouadon et al., 2006] suggesting that JP-45 down regulates the activity of the EC coupling machinery; (3) ablation of JP-45 leads to loss of skeletal muscle strength in three months old mice. Interestingly, in an MH mouse animal model (RYR1_Y522S) Andronache et al. (2009) showed voltage dependence of inactivation of DHPR-mediated Ca²⁺ release were shifted to more negative potentials, indicating that the mice had activated compensatory mechanisms acting on the DHPR to counterbalance the activation of Ca²⁺ release via mutated RyR1 at more negative membrane potentials. We believe that the expression of JP-45 variants represents an additional mechanism counteracting Ca²⁺ release via the RyR1 channel. Indeed in myotubes and reconstituted FDB fibers peak Ca²⁺ release was similar in cells carrying WT or JP-45 variants, but the EC₅₀ for depolarization-induced Ca²⁺ release was shifted by about 20 mM KCl to higher KCl concentrations. Similarly, electrophysiological measurements of fibers reconstituted with JP-45 variants showed that half of the maximal charge movement and Ca²⁺ currents were recorded at more positive potentials than in fibers reconstituted with wild type JP-45.

Figure 1 shows the human and mouse JP-45 amino acid sequences and the location of the polymorphic variants in a cartoon model. The p.P108L variant falls within a stretch of conserved proline residues which are present in the NH₂ domain of JP-45; substitution of the helix breaking proline residue for the hydrophobic amino acid leucine may change the structure of the domains interacting with the Ca_v1.1 subunit of the DHPR leading to the observed desensitization of the voltage sensor. On the other hand, the effect of the more common p.G150A, which lies right after the predicted transmembrane domain is more difficult to envisage as both glycine and alanine are neutral nonpolar residues, have a similar isoelectric point (5.95 and 6.0, respectively) but differ in the size of their side chains [Voet and Voet, 2004]. Thus the subsitution of the H⁺ side chain on glycine for the CH₃ side group on alanine immediately after the hydrophobic domain may cause conformational changes and/or affect posttranscriptional modifications.

In conclusion we show that the presence of genetic variants of JP-45 affects the function of the voltage sensing DHPR suggesting that variants of other proteins present in the SR interacting with the RyR1 and /or with the DHPR may similarly affect EC coupling and thus will impact not only the severity of an individual's MH phenotype but potentially also the severity of neuromuscular disorders particularly of the core myopathies.

ACKNOWLEDGMENTS

We wish to thank Anne-Sylvie Monnet and Martine Singer for expert technical assistance.