Genetic Analysis of Voltage-Dependent Calcium Channels

Molecular cloning of calcium channel subunit genes has identified an unexpectedly large number of genes and splicing variants, and a central problem of calcium channel biology is to now understand the functional significance of this genetic complexity. While electrophyisological, pharmacological, and molecular cloning techniques are providing one level of understanding, a complete understanding will require many additional kinds of studies, including genetic studies done in intact animals. In this regard, an intriguing variety of episodic diseases have recently been identified that result from defects in calcium channel genes. A study of these diseases illustrates the kind of insights into calcium channel function that can be expected from this method of inquiry.     

Malignant-Hyperthermia Susceptibility Is Associated with a Mutation of the a1-Subunit of the Human Dihydropyridine-Sensitive L-Type Voltage-Dependent Calcium-Channel Receptor in Skeletal Muscle

Malignant hyperthermia susceptibility (MHS) is characterized by genetic heterogeneity. However, except for the MHS1 locus, which corresponds to the skeletal muscle ryanodine receptor (RYR1) and for which several mutations have been described, no direct molecular evidence for a mutation in another gene has been reported so far. In this study we show that the CACNL1A3 gene encoding the alpha 1-subunit of the human skeletal muscle dihydropyridine-sensitive L-type voltage-dependent calcium channel (VDCC) represents a new MHS locus and is responsible for the disease in a large French family. Linkage analysis performed with an intragenic polymorphic microsatellite marker of the CACLN1A3 gene generated a two-point LOD score of 4.38 at a recombinant fraction of 0. Sequence analysis of the coding region of the CACLN1A3 gene showed the presence of an Arg-His substitution at residue 1086, resulting from the transition of A for G3333, which segregates perfectly with the MHS phenotype in the family. The mutation is localized in a very different part of the alpha 1-subunit of the human skeletal muscle VDCC, compared with previously reported mutations found in patients with hypokalemic periodic paralysis, and these two diseases might be discussed in terms of allelic diseases. This report is the first direct evidence that the skeletal muscle VDCC is involved in MHS, and it suggests a direct interaction between the skeletal muscle VDCC and the ryanodine receptor in the skeletal muscle sarcoplasmic reticulum.     

Biochemical analysis of L-type calcium channels from skeletal and cardiac muscle

The development of specific pharmacological agents that modulate different types of ion channels has prompted an extensive effort to elucidate the molecular structure of these important molecules. The calcium channel blockers that specifically modulate the L-type calcium channel activity have aided in the purification and reconstitution of this channel from skeletal muscle transverse tubules. The L-type calcium channel from skeletal muscle is composed of five subunits designated alpha 1, alpha 2, beta, gamma, and sigma. The alpha 1-subunit is the pore-forming polypeptide and contains the ligand binding and phosphorylation sites through which channel activity can be modulated. The role of the other subunits in channel function remains to be studied. The calcium channel components have also been partially purified from cardiac muscle. The channel consists of at least three subunits that have properties related to the subunits of the calcium channel from skeletal muscle. A core polypeptide that can form a channel and contains ligand binding and phosphorylation sites has been identified in cardiac preparations. Here we summarize recent biochemical and molecular studies describing the structural features of these important ion channels.     

Retrospective study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Guthrie cards from a large cohort of neonatal screening for cystic fibrosis

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride channel, and in individuals with both alleles of the gene mutated, symptoms of CF disease are manifest. With more than 300 mutations so far described in the gene the profile of mutant alleles in a population is specific to its ethnic origin. For an analysis with an unbiased recruitment of the CF alleles in neonates of similar origin (Normandy, France), we have retrospectively analyzed the Guthrie cards of affected newborns, diagnosed by the immunoreactive trypsinogen (IRT) assay. Analysis of the 27 exons of the CFTR gene using a GC clamp denaturing gradient gel electrophoresis (DGGE) assay has enabled us to identify over 96% of the mutated alleles. Two of these were novel mutations. We would like to propose this strategy as an efficient method of retrospective molecular genetic diagnosis that can be performed wherever Guthrie cards can be obtained. Knowledge of rare alleles could be a prerequisite for CF therapy in the future.     

Genetic analysis of a synaptic calcium channel in Drosophila: intragenic modifiers of a temperature-sensitive paralytic mutant of cacophony

Our previous genetic analysis of synaptic mechanisms in Drosophila identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel alpha1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cac(TS2), indicated cac encodes a primary calcium channel alpha1 subunit functioning in neurotransmitter release. To further examine the functions and interactions of cac-encoded calcium channels, a genetic screen was performed to isolate new mutations that modify the cac(TS2) paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cac(TS2), including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Here we report molecular characterization of three intragenic modifiers and examine the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs. These mutations may further define the structural basis of calcium channel alpha1 subunit function in neurotransmitter release.     

Genetic modifiers of the Drosophila NSF mutant, comatose, include a temperature-sensitive paralytic allele of the calcium channel alpha1-subunit gene, cacophony

The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Our previous work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1 function and to broaden our analysis of synaptic transmission to other gene products, we have performed a genetic screen for mutations that interact with comt. Here we report the isolation and analysis of four mutations that modify TS paralysis in comt, including two intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel alpha1-subunit gene, cacophony (cac). Analysis of synaptic function in these mutants alone and in combination will further define the in vivo functions and interactions of specific gene products in synaptic transmission.     

Role of P/Q calcium channel in familial hemiplegic migraine

Voltage-dependent calcium channels constitute one of the main pathways of calcium entry into neurons. They are the principal actors of synaptic transmission by controlling the release of neurotransmitters. They also contribute to numerous other cell functions, such as gene expression or synaptogenesis. These channels, by their essential cell functions, are at the origin of numerous channelopathies resulting from mutations of the genes encoding their different subunits. Familial Hemiplegic Migraine (FHM) represents one such example of these channelopathies. In this human disease, genetic studies have demonstrated the implication of the CACNA1A gene in a type 1 form of FHM. This gene encodes for the Ca(v)2.1 subunit of P/Q calcium channels and is the target of numerous mutations affecting the properties of channel activity. The question on how discrete mutations of this gene are able to alter the activity of the channel and contribute to the physiopathology of FHM remains an open question. The functional characterization of mutated channels in various heterologous expression systems, as well as in vivo in an animal model, provides a molecular scheme of the physiopathology of FHM in which neurons, astrocytes and blood circulation act in concert.     

Evidence for the existence of a cardiac specific isoform of the alpha 1 subunit of the voltage dependent calcium channel

Biochemical, pharmacological and electrophysiological evidence implies the existence of tissue specific isoforms of the L-type VDCC. The alpha 1 and alpha 2 subunits of the skeletal muscle calcium channel have been previously cloned and their amino acid sequence deduced. Here we report the isolation and sequencing of a partial cDNA that encodes a heart specific isoform of the alpha 1 subunit. The amino acid sequence deduced from this part cDNA clone shows 64.7% similarity with the skeletal muscle alpha 1 subunit. Northern analysis reveals 2 hybridizing bands, 8.5 and 13 kb, in contrast to one 6.5 kb band in the skeletal muscle. Selective inhibition of mRNA expression in Xenopus oocytes by complementary oligodeoxy-nucleotides derived from the heart clone provides further evidence that the cDNA corresponds to an essential component of the VDCC. These data further support the existence of tissue-specific isoforms of the L-type VDCC.     

Nonchannel Functions of the Calcium Channel γ Subunit: Insight from Research on the Stargazer Mutant

Voltage-dependent calcium channels (VDCC) are essential regulators of intracellular calcium concentration, which in turn influences a broad spectrum of cellular functions especially in neurons. Identification of several calcium channel mutations as the cause of neurological disorders in human and mouse indicates the importance of the integrity of these channels to neuronal function. Studies of mutant mice, each carrying a disrupted gene of a different VDCC subunit, have revealed many unexpected roles of these molecules and have significantly advanced our knowledge of subunit function in the last few years. This review addresses recent discoveries of the function of the gamma2 subunit, also named stargazin, with special emphasis on roles other than calcium conductance.     

Molecular Lesions Associated with Alleles of Decapentaplegic Identify Residues Necessary for Tgf-β/Bmp Cell Signaling in Drosophila Melanogaster

We have identified the molecular lesions associated with six point mutations in the Drosophila TGF-β homologue decapentaplegic (dpp). The sites of these mutations define residues within both the pro and ligand regions that are essential for dpp function in vivo. While all of these mutations affect residues that are highly conserved among TGF-β superfamily members, the phenotypic consequences of the different alleles are quite distinct. Through an analysis of these mutant phenotypes, both in cuticle preparations and with molecular probes, we have assessed the functional significance of specific residues that are conserved among the different members of the superfamily. In addition, we have tested for conditional genetic interactions between the different alleles. We show that two of the alleles are temperature sensitive for the embyronic functions of dpp, such that these alleles are not only embryonic viable as homozygotes but also partially complement other dpp hypomorphs at low temperatures. Our results are discussed with regard to in vitro mutagenesis data on other TGF-β-like molecules, as well as with regard to the regulation of dpp cell signaling in Drosophila.     

Learning from stargazin: the mouse, the phenotype and the unexpected

The stargazin gene (also referred to as Cacng2) has been identified by forward genetics in a spontaneous mouse mutant with ataxic gait, upward head-elevating movements (hence the name stargazer for the mouse) and episodes of spike-wave discharges. Stargazin is related to the gamma-1 subunit of skeletal muscle voltage-dependent calcium channel (VDCC), and a deficit in its role as auxiliary VDCC subunit was proposed to underlie the epileptic phenotype of the mouse; yet, a conclusive demonstration of stargazin function in VDCC regulation is still lacking. In contrast, stargazin and its three closely related isoforms gamma-3, gamma-4 and gamma-8 were shown to function as auxiliary subunits for a very different ion channel - the AMPA-type glutamate receptor - prominently regulating early intracellular transport, synaptic targeting and anchoring, and ion channel functions of this major excitatory receptor in the brain.     

The Unc-8 and Sup-40 Genes Regulate Ion Channel Function in Caenorhabditis Elegans Motorneurons

Two Caenorhabditis elegans genes, unc-8 and sup-40, have been newly identified, by genetic criteria, as regulating ion channel function in motorneurons. Two dominant unc-8 alleles cause motorneuron swelling similar to that of other neuronal types in dominant mutants of the deg-1 gene family, which is homologous to a mammalian gene family encoding amiloride-sensitive sodium channel subunits. As for previously identified deg-1 family members, unc-8 dominant mutations are recessively suppressed by mutations in the mec-6 gene, which probably encodes a second type of channel component. An unusual dominant mutation, sup-41 (lb125), also co-suppresses unc-8 and deg-1, suggesting the existence of yet another common component of ion channels containing unc-8 or deg-1 subunits. Dominant, transacting, intragenic suppressor mutations have been isolated for both unc-8 and deg-1, consistent with the idea that, like their mammalian homologues, the two gene products function as multimers. The sup-40 (lb130) mutation dominantly suppresses unc-8 motorneuron swelling and produces a novel swelling phenotype in hypodermal nuclei. sup-40 may encode an ion channel component or regulator that can correct the osmotic defect caused by abnormal unc-8 channels.     

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.     

Mouse models and the interpretation of human GWAS in type 2 diabetes and obesity

Within the last 3 years, genome-wide association studies (GWAS) have had unprecedented success in identifying loci that are involved in common diseases. For example, more than 35 susceptibility loci have been identified for type 2 diabetes and 32 for obesity thus far. However, the causal gene and variant at a specific linkage disequilibrium block is often unclear. Using a combination of different mouse alleles, we can greatly facilitate the understanding of which candidate gene at a particular disease locus is associated with the disease in humans, and also provide functional analysis of variants through an allelic series, including analysis of hypomorph and hypermorph point mutations, and knockout and overexpression alleles. The phenotyping of these alleles for specific traits of interest, in combination with the functional analysis of the genetic variants, may reveal the molecular and cellular mechanism of action of these disease variants, and ultimately lead to the identification of novel therapeutic strategies for common human diseases. In this Commentary, we discuss the progress of GWAS in identifying common disease loci for metabolic disease, and the use of the mouse as a model to confirm candidate genes and provide mechanistic insights.     

The genetic and molecular basis of epilepsy

In the past decade, studies of large families in which epilepsy has been inherited in an autosomal dominant fashion have revealed several mutated genes, most of which encode ion channel subunits. Despite these exciting findings, only a few families with similar phenotypes have mutations in these known genes. More frustrating has been the genetic research into idiopathic epilepsies with complex inheritance. Although these forms are more common than those with Mendelian inheritance, their unknown mode of inheritance, phenotypic heterogeneity and the uncertainty of the genetic overlap among syndrome subtypes have hampered gene mapping. New techniques of molecular analysis could help the dissection of genes for epilepsies with complex inheritance. Hopefully, in the near future, successful genetic studies will make possible the discovery of new and more-targeted anti-epileptic drugs.     

Understanding anthelmintic resistance: the need for genomics and genetics

Anthelmintic resistance is a major problem for the control of many parasitic nematode species and has become a major constraint to livestock production in many parts of the world. In spite of its increasing importance, there is still a poor understanding of the molecular and genetic basis of resistance. It is unclear which mutations contribute most to the resistance phenotype and how resistance alleles arise, are selected and spread in parasite populations. The main strategy used to identify mutations responsible for anthelmintic resistance has been to undertake experimental studies on candidate genes. These genes have been chosen predominantly on the basis of our knowledge of drug mode-of-action and the identification of mutations that can confer resistance in model organisms. The application of these approaches to the analysis of benzimidazole and ivermectin resistance is reviewed and the reasons for their relative success or failure are discussed. The inherent limitation of candidate gene studies is that they rely on very specific and narrow assumptions about the likely identity of resistance-associated genes. In contrast, forward genetic and functional genomic approaches do not make such assumptions, as illustrated by the successful application of these techniques in the study of insecticide resistance. Although there is an urgent need to apply these powerful approaches to anthelmintic resistance research, the basic methodologies and resources are still lacking. However, these are now being developed for the trichostrongylid nematode Haemonchus contortus and the current progress and research priorities in this area are discussed.     

The Role of Auxiliary Subunits for the Functional Diversity of Voltage‐Gated Calcium Channels

Voltage‐gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore‐forming α₁ subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice‐variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre‐existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc.     

Calcium channels and channelopathies of the central nervous system

Several inherited human neurological disorders can be caused by mutations in genes encoding Ca²⁺ channel subunits. This review deals with known human and mouse calcium channelopathies of the central nervous system (CNS). The human diseases comprise: 1) a recessive retinal disorder, X-linked congenital stationary night blindness, associated with mutations in the CACNA1F gene, encoding α₁1.4 subunits of L-type channels; and 2) a group of rare allelic autosomal dominant human neurological disorders including familial hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia type 6, all associated with mutations in the CACNA1A gene, encoding α₁2.1 subunits of P/Q-type calcium channels. Mutations at the mouse orthologue of the CACNA1A gene cause a group of recessive neurological disorders, including the tottering, leaner, and rocker phenotypes with ataxia and absence epilepsy, and the rolling Nagoya phenotype with ataxia without seizures. Two other spontaneous mouse mutants with ataxia and absence epilepsy, lethargic and stargazer, have mutations in genes encoding a calcium channel auxiliary β subunit and a putative calcium channel auxiliary γ subunit. For each channelopathy, the review describes disease phenotype, channel genotype, and known functional consequences of the pathological mutations; in some cases, it also describes working hypothesis and/or speculations addressing the challenging question of how the alterations in channel function lead to selective cellular dysfunction and disease.     

Molecular analysis of Drosophila eyes absent mutants reveals features of the conserved Eya domain

The eyes absent (eya) gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and physically interacts in vitro with two other genes of eye development, sine oculis and dachshund. The Eya protein sequence, while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, we have sequenced eya alleles. These mutations have revealed that loss of the entire Eya Domain is null for eya activity, but that alleles with truncations within the Eya Domain display partial function. We then extended the molecular genetic analysis to interactions within the Eya Domain. This analysis has revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminished the interactions with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions within it.     

Functional expression of human and Arabidopsis protein phosphatase 2A in Saccharomyces cerevisiae and isolation of dominant-defective mutants.

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic subunit and two distinct regulatory subunits, A and B. The primary sequence of the catalytic (C) subunit is highly conserved in evolution, and its function has been shown to be essential in yeast, Drosophila and mice. In many eukaryotes, the C subunit is encoded by at least two nearly identical genes, impeding conventional loss-of-function genetic analysis. We report here the development of a functional complementation assay in S. cerevisiae that has allowed us to isolate dominant-defective alleles of human and Arabidopsis C subunit genes. Wild-type human and Arabidopsis C subunit genes can complement the lethal phenotype of S. cerevisiae PP2A-C mutations. Site-directed mutagenesis was used to create two distinct, catalytically impaired C subunit mutants of the human and Arabidopsis genes. In both cases, expression of the mutant subunit in yeast prevented growth, even in the presence of functional C subunit proteins. This dominant growth defect is consistent with a dominant-interfering mode of action. Thus, we have shown that S. cerevisiae provides a rapid system for the functional analysis of heterologous PP2A genes, and that two mutations that abrogate phosphatase activity exhibit dominant-defective phenotypes in S. cerevisiae.