Temperature and RyR1 Regulate the Activation Rate of Store-Operated Ca(2+) Entry Current in Myotubes

Store-operated calcium entry (SOCE) is an important Ca²⁺ entry pathway in skeletal muscle. However, direct electrophysiological recording and full characterization of the underlying SOCE current in skeletal muscle cells (I_SkCRAC) has not been reported. Here, we characterized the biophysical properties, pharmacological profile, and molecular identity of I_SkCRAC in skeletal myotubes, as well as the regulation of its rate of activation by temperature and the type I ryanodine receptor (RyR1). I_SkCRAC exhibited many hallmarks of Ca²⁺ release activated Ca²⁺ currents (I_CRAC): store dependence, strong inward rectification, positive reversal potential, limited cesium permeability, and sensitivity to SOCE channel blockers. I_SkCRAC was reduced by siRNA knockdown of stromal interaction molecule 1 and expression of dominant negative Orai1. Average I_SkCRAC current density at −80mV was 1.00 ± 0.05 pA/pF. In the presence of 20 mM intracellular EGTA, I_SkCRAC activation occurred over tens of seconds during repetitive depolarization at 0.5Hz and was inhibited by treatment with 100 μM ryanodine. The rate of SOCE activation was reduced threefold in myotubes from RyR1-null mice and increased 4.6-fold at physiological temperatures (35–37°C). These results show that I_SkCRAC exhibits similar biophysical, pharmacological, and molecular properties as I_CRAC in nonexcitable cells and its rate of activation during repetitive depolarization is strongly regulated by temperature and RyR1 activity.     

Allele-Specific Gene Silencing in Two Mouse Models of Autosomal Dominant Skeletal Myopathy

We explored the potential of mutant allele-specific gene silencing (ASGS) in providing therapeutic benefit in two established mouse models of the autosomal dominantly-inherited muscle disorders, Malignant Hyperthermia (MH) and Central Core Disease (CCD). Candidate ASGS siRNAs were designed and validated for efficacy and specificity on ryanodine receptor (RyR1) cDNA mini-constructs expressed in HEK293 cells using RT-PCR- and confocal microscopy-based assays. In vivo delivery of the most efficacious identified siRNAs into flexor digitorum brevis (FDB) muscles was achieved by injection/electroporation of footpads of 4–6 month old heterozygous Ryr1^Y524S/+ (YS/+) and Ryr1^I4895T/+ (IT/+) knock-in mice, established mouse models of MH with cores and CCD, respectively. Treatment of IT/+ mice resulted in a modest rescue of deficits in the maximum rate (∼38% rescue) and magnitude (∼78%) of ligand-induced Ca²⁺ release that occurred in the absence of a change in the magnitude of electrically-evoked Ca²⁺ release. Compared to the difference between the caffeine sensitivity of Ca²⁺ release in FDB fibers from YS/+ and WT mice treated with SCR siRNA (EC₅₀: 1.1 mM versus 4.4 mM, respectively), caffeine sensitivity was normalized in FDB fibers from YS/+ mice following 2 (EC₅₀: 2.8 mM) and 4 week (EC₅₀: 6.6 mM) treatment with YS allele-specific siRNA. Moreover, the temperature-dependent increase in resting Ca²⁺ observed in FDB fibers from YS/+ mice was normalized to WT levels after 2 weeks of treatment with YS allele-specific siRNA. As determined by quantitative real time PCR, the degree of functional rescue in YS/+ and IT/+ mice correlated well with the relative increase in fractional WT allele expression.     

Heteromeric TRPV4-C1 channels contribute to store-operated Ca(2+) entry in vascular endothelial cells

There is controversy as to whether TRP channels participate in mediating store-operated current (I_SOC) and store-operated Ca²⁺ entry (SOCE). Our recent study has demonstrated that TRPC1 forms heteromeric channels with TRPV4 in vascular endothelial cells and that Ca²⁺ store depletion enhances the vesicle trafficking of heteromeric TRPV4-C1 channels, causing insertion of more channels into the plasma membrane in vascular endothelial cells. In the present study, we determined whether the enhanced TRPV4-C1 insertion to the plasma membrane could contribute to SOCE and I_SOC. We found that thapsigargin-induced SOCE was much lower in aortic endothelial cells derived from trpv4^−/− or trpc1^−/− knockout mice when compared to that of wild-type mice. In human umbilical vein endothelial cells (HUVECs), thapsigargin-induced SOCE was markedly reduced by knocking down the expression of TRPC1 and/or TRPV4 with respective siRNAs. Brefeldin A, a blocker of vesicular translocation, inhibited the SOCE. These results suggest that an enhanced vesicular trafficking of heteromeric TRPV4-C1 channels contributes to SOCE in vascular endothelial cells. Vascular tension studies suggest that such an enhanced trafficking of TRPV4-C1 channels may play a role in thapsigargin-induced vascular relaxation in rat small mesenteric arteries.     

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions

Store-operated Ca²⁺ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²⁺ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I_SOC, activated in response to Ca²⁺ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I_CRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(⁶⁸⁴EE⁶⁸⁵). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²⁺ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³⁺, removal of extracellular Ca²⁺, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²⁺-containing, but not Ca²⁺-free, medium. Consistent with this, I_CRAC is activated in cells pretreated with thapsigargin in Ca²⁺-free medium while I_SOC is activated in cells pretreated in Ca²⁺-containing medium. Significantly, TRPC1 function is required for sustained K_Ca activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²⁺ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²⁺ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.     

Increased Store-Operated Ca Entry in Skeletal Muscle with Reduced Calsequestrin-1 Expression

Store-operated Ca²⁺ entry (SOCE) contributes to Ca²⁺ handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn²⁺ fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca²⁺ store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39°C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca²⁺ permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca²⁺ as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca²⁺ entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca²⁺ signaling in MH.     

The pore region of the skeletal muscle ryanodine receptor is a primary locus for excitation-contraction uncoupling in central core disease

Human central core disease (CCD) is caused by mutations/deletions in the gene that encodes the skeletal muscle ryanodine receptor (RyR1). Previous studies have shown that CCD mutations in the NH2-terminal region of RyR1 lead to the formation of leaky SR Ca2+ release channels when expressed in myotubes derived from RyR1-knockout (dyspedic) mice, whereas a COOH-terminal mutant (I4897T) results in channels that are not leaky to Ca2+ but lack depolarization-induced Ca2+ release (termed excitation-contraction [EC] uncoupling). We show here that store depletion resulting from NH2-terminal (Y523S) and COOH-terminal (Y4795C) leaky CCD mutant release channels is eliminated after incorporation of the I4897T mutation into the channel (Y523S/I4897T and Y4795C/I4897T). In spite of normal SR Ca2+ content, myotubes expressing the double mutants lacked voltage-gated Ca2+ release and thus exhibited an EC uncoupling phenotype similar to that of I4897T-expressing myotubes. We also show that dyspedic myotubes expressing each of seven recently identified CCD mutations located in exon 102 of the RyR1 gene (G4890R, R4892W, I4897T, G4898E, G4898R, A4905V, R4913G) behave as EC-uncoupled release channels. Interestingly, voltage-gated Ca2+ release was nearly abolished (reduced approximately 90%) while caffeine-induced Ca2+ release was only marginally reduced in R4892W-expressing myotubes, indicating that this mutation preferentially disrupts voltage-sensor activation of release. These data demonstrate that CCD mutations in exon 102 disrupt release channel permeation to Ca2+ during EC coupling and that this region represents a primary molecular locus for EC uncoupling in CCD.     

Distinct effects on Ca2+ handling caused by malignant hyperthermia and central core disease mutations in RyR1

Malignant hyperthermia (MH) and central core disease (CCD) are disorders of skeletal muscle Ca2+ homeostasis that are linked to mutations in the type 1 ryanodine receptor (RyR1). Certain RyR1 mutations result in an MH-selective phenotype (MH-only), whereas others result in a mixed phenotype (MH + CCD). We characterized effects on Ca2+ handling and excitation-contraction (EC) coupling of MH-only and MH + CCD mutations in RyR1 after expression in skeletal myotubes derived from RyR1-null (dyspedic) mice. Compared to wild-type RyR1-expressing myotubes, MH + CCD- and MH-only-expressing myotubes exhibited voltage-gated Ca2+ release (VGCR) that activated at more negative potentials and displayed a significantly higher incidence of spontaneous Ca2+ oscillations. However, maximal VGCR was reduced only for MH + CCD mutants (Y4795C, R2435L, and R2163H) in which spontaneous Ca2+ oscillations occurred with significantly longer duration (Y4795C and R2435L) or higher frequency (R2163H). Notably, myotubes expressing these MH + CCD mutations in RyR1 exhibited both increased [Ca2+]i and reduced sarcoplasmic reticulum (SR) Ca2+ content. We conclude that MH-only mutations modestly increase basal release-channel activity in a manner insufficient to alter net SR Ca2+ content ("compensated leak"), whereas the mixed MH + CCD phenotype arises from mutations that enhance basal activity to a level sufficient to promote SR Ca2+ depletion, elevate [Ca2+]i, and reduce maximal VGCR ("decompensated leak").     

Retigabine,a Kv7.2/Kv7.3-Channel Opener,Attenuates Drug-Induced Seizures in Knock-In Mice Harboring Kcnq2 Mutations

The hetero-tetrameric voltage-gated potassium channel K_v7.2/K_v7.3, which is encoded by KCNQ2 and KCNQ3, plays an important role in limiting network excitability in the neonatal brain. K_v7.2/K_v7.3 dysfunction resulting from KCNQ2 mutations predominantly causes self-limited or benign epilepsy in neonates, but also causes early onset epileptic encephalopathy. Retigabine (RTG), a K_v7.2/ K_v7.3-channel opener, seems to be a rational antiepileptic drug for epilepsies caused by KCNQ2 mutations. We therefore evaluated the effects of RTG on seizures in two strains of knock-in mice harboring different Kcnq2 mutations, in comparison to the effects of phenobarbital (PB), which is the first-line antiepileptic drug for seizures in neonates. The subjects were heterozygous knock-in mice (Kcnq2^Y284C/+ and Kcnq2^A306T/+) bearing the Y284C or A306T Kcnq2 mutation, respectively, and their wild-type (WT) littermates, at 63–100 days of age. Seizures induced by intraperitoneal injection of kainic acid (KA, 12mg/kg) were recorded using a video-electroencephalography (EEG) monitoring system. Effects of RTG on KA-induced seizures of both strains of knock-in mice were assessed using seizure scores from a modified Racine’s scale and compared with those of PB. The number and total duration of spike bursts on EEG and behaviors monitored by video recording were also used to evaluate the effects of RTG and PB. Both Kcnq2^Y284C/+ and Kcnq2^A306T/+ mice showed significantly more KA-induced seizures than WT mice. RTG significantly attenuated KA-induced seizure activities in both Kcnq2^Y284C/+ and Kcnq2^A306T/+ mice, and more markedly than PB. This is the first reported evidence of RTG ameliorating KA-induced seizures in knock-in mice bearing mutations of Kcnq2, with more marked effects than those observed with PB. RTG or other K_v7.2-channel openers may be considered as first-line antiepileptic treatments for epilepsies resulting from KCNQ2 mutations.     

Enhanced excitation-coupled calcium entry in myotubes is associated with expression of RyR1 malignant hyperthermia mutations

Myotubes expressing wild type RyR1 (WT) or RyR1 with one of three malignant hyperthermia mutations R615C, R2163C, and T4826I (MH) were exposed sequentially to 60 mm KCl in Ca(2+)-replete and Ca(2+)-free external buffers (Ca+ and Ca-, respectively) with 3 min of rest between exposures. Although the maximal peak amplitude of the Ca(2+) transients during K(+) depolarization was similar for WT and MH in both external buffers, the rate of decay of the sustained phase of the transient during K(+) depolarization (decay rate) in Ca+ was 50% slower for MH. This difference was eliminated in Ca-, and the relative decay rates were faster for both genotypes than in Ca+. The integrated Ca(2+) transient in Ca-compared with Ca+ was reduced by 50-60% for MH and 20% for WT. The decay rate was not affected by [K(+)] x [Cl(-)] product or NiCl(2) (2 mm) supplementation of Ca-. The addition of La(2+) (0.1 mm), or SKF 96365 (20 microm) to Ca+ significantly accelerated decay rates for both WT and MH, but their effect was significantly greater in MH. Nifedipine (1 microm) had no effect, suggesting that the mechanism for this difference was not a reduction in L-type Ca(2+) channel Ca(2+) current. These data strongly suggest: 1) the decay rate in skeletal myotubes is related in part to Ca(2+) entry through the ECCE channel; 2) the MH mutations enhance ECCE compared with wild type; and 3) the increased Ca(2+) entry might play a significant role in the pathophysiology of MH.     

Up-regulation of BMP2/4 signaling increases both osteoblast-specific marker expression and bone marrow adipogenesis in Gja1Jrt/+ stromal cell cultures

Gja1^Jrt/+ mice carry a mutation in one allele of the gap junction protein α1 gene (Gja1), resulting in a G60S connexin 43 (Cx43) mutant protein that is dominant negative for Cx43 protein production of <50% of wild-type (WT) levels and significantly reduced gap junction formation and function in osteoblasts and other Cx43-expressing cells. Previously we reported that Gja1^Jrt/+ mice exhibited early-onset osteopenia caused by activation of osteoclasts secondary to activation of osteoblast lineage cells, which expressed increased RANKL and produced an abnormal resorption-stimulating bone matrix high in BSP content. Gja1^Jrt/+ mice also displayed early and progressive bone marrow atrophy, with a significant increase in bone marrow adiposity versus WT littermates but no increase in adipose tissues elsewhere in the body. BMP2/4 production and signaling were increased in Gja1^Jrt/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1^Jrt/+ osteoblasts and increased Pparg2 expression in bone marrow–derived adipoprogenitors in vitro. The elevated levels of BMP2/4 signaling in G60S Cx43-containing cells resulted at least in part from elevated levels of cAMP. We conclude that up-regulation of BMP2/4 signaling in trabecular bone and/or stromal cells increases osteoblast-specific marker expression in hyperactive Gja1^Jrt/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1^Jrt/+ mouse model.     

Functional interactions among STIM1, Orai1 and TRPC1 on the activation of SOCs in HL-7702 cells

STIM1, Orai1 and TRPC1 are all reported to be important for store-operated Ca²⁺ entry (SOCE) in diverse cells. However, there is no evidence for the functional interaction of the three proteins in SOCE in human liver cells. The objective of this study is to determine whether they are involved in SOCE in normal human liver cells. Liposomal transfection method was used to increase expression levels of the three proteins in HL-7702 cells, a normal human liver cell line. Western blot and single cell RT–PCR were applied to evaluate transfection effectiveness. Changes in store-operated current (I_SOC) and SOCE were investigated using whole-cell patch-clamp recording and calcium imaging. I_SOC is detected in HL-7702 cells and it is inhibited either by 2-Aminoethoxydiphenyl borate (2-APB) or La³⁺. Overexpression of STIM1 or Orai1 alone did not induce any change in I_SOC. TRPC1-transfection, however, caused approximate 2.5-fold increase in I_SOC. A large increase (>10-fold) in I_SOC emerged when both STIM1 and Orai1 were co-transfected into HL-7702 cells. Co-overexpression of STIM1 + TRPC1 also caused >10-fold increase in I_SOC, and addition of Orai1 did not cause any further increase. In HL-7702 cells, TRPC1 and Orai1 take part in SOCE independently of each other. Functional interactions of STIM1 and Orai1 or TRPC1 contribute to I_SOC activation.     

Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The Apc^Min/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, Apc^Min/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc^Min/+ WT and Apc^Min/+ TLR4^?/? mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of Apc^Min/+ TLR4^?/? mice compared with Apc^Min/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.     

Hyperpolarization-Activated Current (Ih) Is Reduced in Hippocampal Neurons from Gabra5?/? Mice

Changes in the expression of γ-aminobutyric acid type A (GABA_A) receptors can either drive or mediate homeostatic alterations in neuronal excitability. A homeostatic relationship between α5 subunit-containing GABA_A (α5GABA_A) receptors that generate a tonic inhibitory conductance, and HCN channels that generate a hyperpolarization-activated cation current (I_h) was recently described for cortical neurons, where a reduction in I_h was accompanied by a reciprocal increase in the expression of α5GABA_A receptors resulting in the preservation of dendritosomatic synaptic function. Here, we report that in mice that lack the α5 subunit gene (Gabra5−/−), cultured embryonic hippocampal pyramidal neurons and ex vivo CA1 hippocampal neurons unexpectedly exhibited a decrease in I_h current density (by 40% and 28%, respectively), compared with neurons from wild-type (WT) mice. The resting membrane potential and membrane hyperpolarization induced by blockade of I_h with ZD-7288 were similar in cultured WT and Gabra5−/− neurons. In contrast, membrane hyperpolarization measured after a train of action potentials was lower in Gabra5−/− neurons than in WT neurons. Also, membrane impedance measured in response to low frequency stimulation was greater in cultured Gabra5−/− neurons. Finally, the expression of HCN1 protein that generates I_h was reduced by 41% in the hippocampus of Gabra5−/− mice. These data indicate that loss of a tonic GABAergic inhibitory conductance was followed by a compensatory reduction in I_h. The results further suggest that the maintenance of resting membrane potential is preferentially maintained in mature and immature hippocampal neurons through the homeostatic co-regulation of structurally and biophysically distinct cation and anion channels.     

Glibenclamide and HMR1098 normalize Cantú syndrome‐associated gain‐of‐function currents

Cantú syndrome (CS) is caused by dominant gain‐of‐function mutation in ATP‐dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but I_KATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that I_KATP inhibitors, glibenclamide and HMR1098, normalize CS channels. I_KATP in response to Mg‐ATP, glibenclamide and HMR1098 were measured by inside‐out patch‐clamp electrophysiology. Results were interpreted in view of cryo‐EM I_KATP channel structures. Mg‐ATP IC₅₀ values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild‐type (0.15 ± 0.01). HMR1098 dose‐dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg‐ATP, reaching, at 30 μmol/L, current levels displayed by wild‐type and H60Y channels in the presence of 0.15 mmol/L Mg‐ATP. Glibenclamide (10 μmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg‐ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only.     

The Intracellular Loop of Orai1 Plays a Central Role in Fast Inactivation of Ca2+ Release-activated Ca2+ Channels

Store-operated Ca²⁺ entry (SOCE) due to activation of Ca²⁺ release-activated Ca²⁺ (CRAC) channels leads to sustained elevation of cytoplasmic Ca²⁺ and activation of lymphocytes. CRAC channels consisting of four pore-forming Orai1 subunits are activated by STIM1, an endoplasmic reticulum Ca²⁺ sensor that senses intracellular store depletion and migrates to plasma membrane proximal regions to mediate SOCE. One of the fundamental properties of CRAC channels is their Ca²⁺-dependent fast inactivation. To identify the domains of Orai1 involved in fast inactivation, we have mutated residues in the Orai1 intracellular loop linking transmembrane segment II to III. Mutation of four residues, V¹⁵¹SNV¹⁵⁴, at the center of the loop (MutA) abrogated fast inactivation, leading to increased SOCE as well as higher CRAC currents. Point mutation analysis identified five key amino acids, N¹⁵³VHNL¹⁵⁷, that increased SOCE in Orai1 null murine embryonic fibroblasts. Expression or direct application of a peptide comprising the entire intracellular loop or the sequence N¹⁵³VHNL¹⁵⁷ blocked CRAC currents from both wild type (WT) and MutA Orai1. A peptide incorporating the MutA mutations had no blocking effect. Concatenated Orai1 constructs with four MutA monomers exhibited high CRAC currents lacking fast inactivation. Reintroduction of a single WT monomer (MutA-MutA-MutA-WT) was sufficient to fully restore fast inactivation, suggesting that only a single intracellular loop can block the channel. These data suggest that the intracellular loop of Orai1 acts as an inactivation particle, which is stabilized in the ion permeation pathway by the N¹⁵³VHNL¹⁵⁷ residues. These results along with recent reports support a model in which the N terminus and the selectivity filter of Orai1 as well as STIM1 act in concert to regulate the movement of the intracellular loop and evoke fast inactivation.     

Functional defects in six ryanodine receptor isoform-1 (RyR1) mutations associated with malignant hyperthermia and their impact on skeletal excitation-contraction coupling

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that segregates with >60 mutations within the MHS-1 locus on chromosome 19 coding for ryanodine receptor type 1 (RyR1). Although some MHRyR1s have been shown to enhance sensitivity to caffeine and halothane when expressed in non-muscle cells, their influence on EC coupling can only be studied in skeletal myotubes. We therefore expressed WTRyR1, six of the most common human MHRyR1s (R163C, G341R, R614C, R2163C, V2168M, and R2458H), and a newly identified C-terminal mutation (T4826I) in dyspedic myotubes to study their functional defects and how they influence EC coupling. Myotubes expressing any MHRyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing WTRyR1. The hypersensitivity of MH myotubes extended to K+ depolarization. MH myotubes responded to direct channel activators with maximum Ca2+ amplitudes consistently smaller than WT myotubes, whereas the amplitude of their responses to depolarization were consistently larger than WT myotubes. The magnitudes of responses attainable from myotubes expressing MHRyR1s are therefore related to the nature of the stimulus rather than size of the Ca2+ store. The functional changes of MHRyR1s were directly analyzed using [3H]ryanodine binding analysis of isolated myotube membranes. Although none of the MHRyR1s examined significantly altered EC50 for Ca2+ activation, many failed to be completely inhibited by a low Ca2+ (相似文献   

Differential mechanisms of Cantú syndrome–associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel

Cantú syndrome (CS) is a rare disease characterized by congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects. Recent genetic analysis has revealed that the majority of CS patients carry a missense mutation in ABCC9, which codes for the sulfonylurea receptor SUR2. SUR2 subunits couple with Kir6.x, inwardly rectifying potassium pore-forming subunits, to form adenosine triphosphate (ATP)-sensitive potassium (K_ATP) channels, which link cell metabolism to membrane excitability in a variety of tissues including vascular smooth muscle, skeletal muscle, and the heart. The functional consequences of multiple uncharacterized CS mutations remain unclear. Here, we have focused on determining the functional consequences of three documented human CS-associated ABCC9 mutations: human P432L, A478V, and C1043Y. The mutations were engineered in the equivalent position in rat SUR2A (P429L, A475V, and C1039Y), and each was coexpressed with mouse Kir6.2. Using macroscopic rubidium (⁸⁶Rb⁺) efflux assays, we show that K_ATP channels formed with P429L, A475V, or C1039Y mutants enhance K_ATP activity compared with wild-type (WT) channels. We used inside-out patch-clamp electrophysiology to measure channel sensitivity to ATP inhibition and to MgADP activation. For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate that these three CS mutations all lead to overactive K_ATP channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation.     

STIM1 and Orai1: novel targets for vascular diseases?

The past five years have witnessed the discovery of the endoplasmic reticulum calcium (Ca²⁺) sensor STIM1 and the plasma membrane Ca²⁺ channel Orai1 as the bona fide molecular components of the store-operated Ca²⁺ entry (SOCE) and the Ca²⁺ release-activated Ca²⁺ current (I _CRAC). It has been known for two decades that SOCE and I _CRAC are required for lymphocyte activation as evidenced by severe immunodeficient phenotypes in patients lacking I _CRAC. In recent years however, studies have uncovered expression of STIM1 and Orai1 proteins in various tissues and described additional roles for these proteins in physiological functions and pathophysiological conditions. Here, we will summarize novel findings pertaining to the role of STIM1 and Orai1 in the vascular system and discuss their potential use as targets in the therapy of vascular disease.     

Human Muscle Economy Myoblast Differentiation and Excitation-Contraction Coupling Use the Same Molecular Partners,STIM1 and STIM2

Our recent work identified store-operated Ca²⁺ entry (SOCE) as the critical Ca²⁺ source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370–5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca²⁺ mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca²⁺ content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca²⁺-content during repeated KCl-induced Ca²⁺ releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitation-contraction coupling.