Putative malignant hyperthermia mutation CaV1.1-R174W is insufficient to trigger a fulminant response to halothane or confer heat stress intolerance.

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, and HOM) express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.

A skeletal muscle L-type Ca2+ channel with a mutation in the selectivity filter (CaV1.1 E1014K) conducts K.

A glutamate-to-lysine substitution at position 1014 within the selectivity filter of the skeletal muscle L-type Ca2+ channel (CaV1.1) abolishes Ca2+ flux through the channel pore. Mice engineered to exclusively express the mutant channel display accelerated muscle fatigue, changes in muscle composition, and altered metabolism relative to wildtype littermates. By contrast, mice expressing another mutant CaV1.1 channel that is impermeable to Ca2+ (CaV1.1 N617D) have shown no detectable phenotypic differences from wildtype mice to date. The major biophysical difference between the CaV1.1 E1014K and CaV1.1 N617D mutants elucidated thus far is that the former channel conducts robust Na+ and Cs+ currents in patch-clamp experiments, but neither of these monovalent conductances seems to be of relevance in vivo Thus, the basis for the different phenotypes of these mutants has remained enigmatic. We now show that CaV1.1 E1014K readily conducts 1,4-dihydropyridine-sensitive K+ currents at depolarizing test potentials, whereas CaV1.1 N617D does not. Our observations, coupled with a large body of work by others regarding the role of K+ accumulation in muscle fatigue, raise the possibility that the introduction of an additional K+ flux from the myoplasm into the transverse-tubule lumen accelerates the onset of fatigue and precipitates the metabolic changes observed in CaV1.1 E1014K muscle. These results, highlighting an unexpected consequence of a channel mutation, may help define the complex mechanisms underlying skeletal muscle fatigue and related dysfunctions.

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p > 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3-5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca(2+) channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation. METHODS: We recorded L-type currents, charge movements, and myoplasmic Ca(2+) transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1(G93A)). RESULTS: In FDB fibers obtained from "symptomatic" global SOD1(G93A) mice, we observed a substantial reduction of SR Ca(2+) release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1(G93A) fibers when compared to control fibers. Ca(2+) transients were not significantly reduced in similar experiments performed with FDB fibers obtained from "early-symptomatic" SOD1(G93A) mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca(2+) release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1(G93A) expression was restricted to skeletal muscle. CONCLUSIONS: We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1(G93A) mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1(G93A) and skeletal muscle autonomous MLC/SOD1(G93A) mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status.

Rem uncouples excitation-contraction coupling in adult skeletal muscle fibers.

In skeletal muscle, excitation-contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca(2+) channel (Ca(V)1.1) to be communicated to the type 1 ryanodine-sensitive Ca(2+) release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein-protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca(V)1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α(1S) subunit of Ca(V)1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary ß(1a) subunit of Ca(V)1.1 is a conduit for this intermolecular communication. However, a direct role for ß(1a) has been difficult to test because ß(1a) serves two other functions that are prerequisite for conformational coupling between Ca(V)1.1 and RYR1. Specifically, ß(1a) promotes efficient membrane expression of Ca(V)1.1 and facilitates the tetradic ultrastructural arrangement of Ca(V)1.1 channels within plasma membrane-SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established ß subunit-interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca(2+) transients without greatly affecting Ca(V)1.1 targeting, intramembrane gating charge movement, or releasable SR Ca(2+) store content. In contrast, a ß(1a)-binding-deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca(2+) release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca(V)1.1 from RYR1-mediated SR Ca(2+) release via its ability to interact with ß(1a). Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the ß(1a) subunit in skeletal-type EC coupling.

Functional assessment of three Rem residues identified as critical for interactions with Ca(2+) channel ß subunits.

Members of the Rem, Rem2, Rad, Gem/Kir (RGK) family of small GTP-binding proteins inhibit high-voltage-activated (HVA) Ca(2+) channels through interactions with both the principal α1 and the auxiliary ß subunits of the channel complex. Three highly conserved residues of Rem (R200, L227, and H229) have been shown in vitro to be critical for interactions with ß subunits. However, the functional significance of these residues is not known. To investigate the contributions of R200, L227, and H229 to ß subunit-mediated RGK protein-dependent inhibition of HVA channels, we introduced alanine substitutions into all three positions of Venus fluorescent protein-tagged Rem (V-Rem AAA) and made three other V-Rem constructs with an alanine introduced at only one position (V-Rem R200A, V-Rem L227A, and V-Rem H229A). Confocal imaging and immunoblotting demonstrated that each Venus-Rem mutant construct had comparable expression levels to Venus-wild-type Rem when heterologously expressed in tsA201 cells. In electrophysiological experiments, V-Rem AAA failed to inhibit N-type Ca(2+) currents in tsA201 cells coexpressing CaV2.2 α1B, ß3, and α2δ-1 channel subunits. The V-Rem L227A single mutant also failed to reduce N-type currents conducted by coexpressed CaV2.2 channels, a finding consistent with the previous observation that a leucine at position 227 is critical for Rem-ß interactions. Rem-dependent inhibition of CaV2.2 channels was impaired to a much lesser extent by the R200A substitution. In contrast to the earlier work demonstrating that Rem H229A was unable to interact with ß3 subunits in vitro, V-Rem H229A produced nearly complete inhibition of CaV2.2-mediated currents.

RGK protein-mediated impairment of slow depolarization- dependent Ca2+ entry into developing myotubes.

Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (Ca(V)1.1):(1) voltage-sensor for excitation-contraction (EC) coupling, (2) L-type Ca(2+) channel, and (3) voltage-sensor for slow depolarization-dependent Ca(2+) entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of Ca(V)1.1. However, it is not known whether the latter function that has been attributed to Ca(V)1.1 is subject to modulation by RGK proteins. Thus, the purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly developing, persistent Ca(2+) entry that is dependent on the voltage-sensing capability of Ca(V)1.1. As a means to investigate this question, Venus fluorescent protein-fused RGK proteins(V-Rad, V-Rem and V-Gem) were overexpressed in "normal" mouse myotubes. We observed that such overexpression of V-Rad, V-Rem or V-Gem in myotubes caused marked changes in morphology of the cells. As shown previously for YFPRem,both L-type current and EC coupling were also impaired greatly in myotubes expressing either V-Rad or V-Gem. There ductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca(2+) entry. Our observations provide the first evidence of modulation of this enigmatic Ca(2+) entry pathway peculiar to skeletal muscle.

The interaction between mGluR1 and the calcium channel Cav2.1 preserves coupling in the presence of long Homer proteins.

Group I metabotropic glutamate receptors (mGluR1 and 5) are G protein coupled receptors that regulate neuronal activity in a number of ways. Some of the most well studied functions of group I mGluRs, such as initiation of multiple forms of mGluR-dependent long-term depression, require receptor localization near the post-synaptic density (PSD). This localization is in turn dependent on the Homer family of scaffolding proteins which bind to a small motif on the distal C-termini of mGluR1 and 5, localize the receptors near the PSD, strengthen coupling to post-synaptic effectors and simultaneously uncouple the mGluRs from extra-synaptic effectors such as voltage dependent ion channels. Here the selectivity of this uncoupling process was examined by testing the ability of Homer-2b to uncouple mGluR1 from multiple voltage dependent calcium channels including Ca(V2.2) (N-type), Ca(V3.2) (T-type), and Ca(V2.1) (P/Q-type) expressed in rat sympathetic neurons from the superior cervical ganglion (SCG). Of these, only the mGluR1-Ca(V2.1) modulatory pathway was insensitive to Homer-2b expression. Uncoupling from this channel was achieved by co-expression of an mGluR1 C-terminal protein designed to disrupt a previously described direct interaction between these two proteins, suggesting that this interaction allows incorporation of Ca(V2.1) into the mGluR1/Homer signaling complex, thereby preserving modulation in the presence of scaffolding Homer proteins. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Venus fly trap domain of mGluR1 functions as a dominant negative against group I mGluR signaling.

Metabotropic glutamate receptors (mGluRs) form covalently linked homodimers and contain large, N-terminal extracellular ligand binding, "venus fly trap" (VFT) domains. These domains, when expressed separately, are secreted as disulfide linked dimers and can dimerize with full-length receptors. mGluR splice variants have been described that contain only this domain, but the consequences of their interaction on receptor signaling have not been explored. Here it is shown that an mGluR1 mutant containing only the VFT is retained on the cell surface when a full-length receptor is co-expressed. Further, when expressed in rat superior cervical ganglion (SCG) neurons and modulation of native calcium currents is used as an assay for receptor activity, the VFT acts as a dominant negative with respect to mGluR1 signaling. Although full-length mGluR1 and mGluR5 are not known to heterodimerize, the mGluR5 VFT partially occludes mGluR1 signaling and the mGluR1 VFT potently occludes mGluR5 signaling in SCG neurons. In addition, an mGluR1 point mutant, mGluR1 C140G, which cannot covalently dimerize, functions like the wild-type receptor when expressed alone. The C140G mutant is inhibited by the mGluR1 VFT construct but does not retain the mGluR1 VFT on the cell surface, suggesting that the loss of C140 renders the interaction reversible. Finally, a peptide designed to disrupt mGluR1 dimerization reduced signaling through the C140G mutant receptor, but only when applied intracellularly for several hours, indicating that loss of signaling requires disruption of dimerization prior to plasma membrane insertion.

Altered G-protein coupling in an mGluR6 point mutant associated with congenital stationary night blindness.

The highly specialized metabotropic glutamate receptor type 6 (mGluR6) is postsynaptically localized and expressed only in the dendrites of ON bipolar cells. Upon activation of mGluR6 by glutamate released from photoreceptors, a nonselective cation channel is inhibited, causing these cells to hyperpolarize. Mutations in this gene have been implicated in the development of congenital stationary night blindness type 1 (CSNB1). We investigated five known mGluR6 point mutants that lead to CSNB1 to determine the molecular mechanism of each phenotype. In agreement with other studies, four mutants demonstrated trafficking impairment. However, mGluR6 E775K (E781K in humans) suggested no trafficking or signaling deficiencies measured by our initial assays. Most importantly, our results indicate a switch in G-protein coupling, in which E775K loses G(o) coupling but retains coupling to G(i), which may explain the phenotype.

The mGlu(4) receptor allosteric modulator N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide acts as a direct agonist at mGlu(6) receptors.

Effects of the mGlu(4) receptor allosteric modulator N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) were tested on closely related mGlu6 receptors. Modulation of native calcium currents in isolated sympathetic neurons from rat superior cervical ganglion by expressed mGlu(4) and mGlu(6) receptor was used to assay receptor activity. Glutamate concentration-response curves with and without PHCCC confirmed that the drug is an allosteric modulator of mGlu(4) receptor, without direct agonist activity. Conversely, PHCCC directly activates the mGlu(6) receptor and does not enhance activity of glutamate. Therefore, PHCCC is a direct mGlu(6) receptor agonist, but lacks allosteric modulatory properties.