Calcium Channel Family Research Articles

Calmodulin Is the Ca 2+ Sensor for Ca 2+-Dependent Inactivation of L-Type Calcium Channels

Elevated intracellular Ca 2+ triggers inactivation of L-type calcium channels, providing negative Ca 2+ feedback in many cells. Ca 2+ binding to the main α 1C channel subunit has been widely proposed to initiate such Ca 2+-dependent inactivation. Here, we find that overexpression of mutant, Ca 2+-insensitive calmodulin (CaM) ablates Ca 2+-dependent inactivation in a “dominant-negative” manner. This result demonstrates that CaM is the actual Ca 2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca 2+-dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of α 1C. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.

Chromosomal Localization and Genomic Characterization of the Mouse Melastatin Gene (Mlsn1)

We recently described a novel gene, melastatin, whose expression is inversely correlated with melanoma aggressiveness. Chromosomal localization of this gene places it on mouse chromosome 7 and in the 15q13–q14 region of the human genome. Although expression patterns and chromosomal localization in the mouse are consistent with involvement of melastatin mutations in the mouse ruby-eye-2 defect, congenic analysis showed genetic segregation of the two loci. Cloning of the full-length human cDNA revealed a much larger transcript than we had previously identified, corresponding to a 1533-amino-acid protein product with homology to members of the transient receptor potential (Trp) family of calcium channels. The mouse melastatin gene contains 27 exons and spans at least 58 kb of genomic DNA. The promoter region ofMlsn1contains four potential microphthalmia binding sites including an M box, a transcriptional regulatory element unique to genes with a restricted melanocytic expression pattern. A 1-kbPvuII fragment from this region was capable of driving high levels of luciferase expression in B16 melanoma cells.

Distinct calcium channel isoforms mediate parathyroid hormone and chlorothiazide-stimulated calcium entry in transporting epithelial cells.

Some cells express multiple calcium channel isoforms that are likely to have distinct functions. The present study used molecular cloning and antisense techniques to identify calcium channel isoforms mediating calcium entry in mouse distal convoluted tubule (DCT) cells. The DCT is the major site of hormone- and diuretic-regulated calcium transport in the kidney. Cellular calcium absorption involves entry through apical membrane calcium channels that are sensitive to dihydropyridine-type calcium channel antagonists. Partial cDNA clones corresponding to one isoform of the calcium channel alpha1 pore-forming subunit, alpha1C, and one isoform of the calcium channel beta accessory subunit, beta3, were isolated by RT-PCR. Full-length transcripts were detected by Northern blot analysis in immortalized DCT cells. Antisense oligonucleotides complementary to the alpha1C sequence inhibited the rise of intracellular calcium ([Ca2+]i) induced by the thiazide diuretic, chlorothiazide (CTZ), but not that induced by parathyroid hormone (PTH). However, antisense oligonucleotides complementary to the beta3 sequence inhibited both CTZ- and PTH-induced rises of [Ca2+]i. beta3 antisense oligonucleotides also inhibited the membrane hyperpolarization induced by CTZ but not that triggered by PTH. Thus, members of the voltage-gated calcium channel family are expressed in DCT cells, where they are responsible for hormone- and drug-induced calcium uptake. The results suggest that DCT cells contain multiple calcium channels with distinct roles in the regulation of cellular calcium.

A Spectrum of Mutations in the Second Gene for Autosomal Dominant Polycystic Kidney Disease (PKD2)

Recently the second gene for autosomal dominant polycystic kidney disease (ADPKD), located on chromosome 4q21-q22, has been cloned and characterized. The gene encodes an integral membrane protein, polycystin-2, that shows amino acid similarity to the PKD1 gene product and to the family of voltage-activated calcium (and sodium) channels. We have systematically screened the gene for mutations by single-strand conformation-polymorphism analysis in 35 families with the second type of ADPKD and have identified 20 mutations. So far, most mutations found seem to be unique and occur throughout the gene, without any evidence of clustering. In addition to small deletions, insertions, and substitutions leading to premature translation stops, one amino acid substitution and five possible splice-site mutations have been found. These findings suggest that the first step toward cyst formation in PKD2 patients is the loss of one functional copy of polycystin-2.

PKD2 , a Gene for Polycystic Kidney Disease That Encodes an Integral Membrane Protein

A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding domain.

Nickel block of a family of neuronal calcium channels: subtype- and subunit-dependent action at multiple sites.

Nickel ions have been reported to exhibit differential effects on distinct subtypes of voltage-activated calcium channels. To more precisely determine the effects of nickel, we have investigated the action of nickel on four classes of cloned neuronal calcium channels (alpha1A, alpha1B, alpha1C, and alpha1E) transiently expressed in Xenopus oocytes. Nickel caused two major effects: (i) block detected as a reduction of the maximum slope conductance and (ii) a shift in the current-voltage relation towards more depolarized potentials which was paralleled by a decrease in the slope of the activation-curve. Block followed 1:1 kinetics and was most pronounced for alpha1C, followed by alpha1E > alpha1A > alpha1B channels. In contrast, the change in activation-gating was most dramatic with alpha1E, with the remaining channel subtypes significantly less affected. The current-voltage shift was well described by a simple model in which nickel binding to a saturable site resulted in altered gating behavior. The affinity for both the blocking site and the putative gating site were reduced with increasing concentration of external permeant ion. Replacement of barium with calcium reduced both the degree of nickel block and the maximal effect on gating for alpha1A channels, but increased the nickel blocking affinity for alpha1E channels. The coexpression of Ca channel beta subunits was found to differentially influence nickel effects on alpha1A, as coexpression with beta2a or with beta4 resulted in larger current-voltage shifts than those observed in the presence of beta1b, while elimination of the beta subunit almost completely abolished the gating shifts. In contrast, block was similar for the three beta subunits tested, while complete removal of the beta subunit resulted in an increase in blocking affinity. Our data suggest that the effect of nickel on calcium channels is complex, cannot be described by a single site of action, and differs qualitatively and quantitatively among individual subtypes and subunit combinations.

Determinants of PKC-dependent modulation of a family of neuronal calcium channels

The modulation of Ca 2+ channel activity by protein kinases contributes to the dynamic regulation of neuronal physiology. Using the transient expression of a family of neuronal Ca 2+ channels, we have identified several factors that contribute to the PKC-dependent modulation of Ca 2+ channels. First, the nature of the Ca 2+ channel α 1 subunit protein is critical. Both α 1 B α 1 E channels exhibit a 30%–40% increase in peak currents after exposure to phorbol esters, whereas neither α 1 A nor α 1 C channels are significantly affected. This up-regulation can be mimicked for α 1 E channels by stimulation of a coexpressed metabotropic glutamate receptor (type 1α) through a PKC-dependent pathway. Second, PKC-stimulated up-regulation is dependent upon coexpression with a Ca 2+ channel β subunit. Third, substitution of the cytoplasmic domain I–II linker from α 1 B confers PKC sensitivity to α 1 A channels. The results provide direct evidence for the modulation of a subset of neuronal Ca 2+ channels by PKC and implicate α 1 and β subunit interactions in regulating channel activity via second messenger pathways.

Subunit-dependent modulation of recombinant L-type calcium channels. Molecular basis for dihydropyridine tissue selectivity.

At least four calcium channel subtypes (P, T, N, and L) have now been classified on the basis of their biophysical and/or pharmacological properties. L-type channels, a channel family particularly important to physiological function of the cardiovascular system, are identified by their slow voltage- and calcium-dependent inactivation as well as their sensitivity to dihydropyridine (DHP) calcium channel antagonists. In this study, we report the results of experiments in which we have measured the DHP modulation of recombinant calcium channel activity in cells transfected with alpha 1 subunits of cardiac and smooth muscle L-type calcium channels. We find subunit-dependent differences in the voltage and concentration dependence of channel modulation. Our results provide evidence for a molecular basis for DHP sensitivity of heart and smooth muscle calcium channels and, additionally, indicate that, even within one family of calcium channels, slight differences in channel structure can cause marked differences in channel pharmacology.

Structure and Functional Expression of a Member of the Low Voltage-Activated Calcium Channel Family

Oscillatory firing patterns are an intrinsic property of some neurons and have an important function in information processing. In some cells, low voltage-activated calcium channels have been proposed to underlie a depolarizing potential that regulates bursting. The sequence of a rat brain calcium channel alpha 1 subunit (rbE-II) was deduced. Although it is structurally related to high voltage-activated calcium channels, the rbE-II channel transiently activated at negative membrane potentials, required a strong hyperpolarization to deinactivate, and was highly sensitive to block by nickel. In situ hybridization showed that rbE-II messenger RNA is expressed in regions throughout the central nervous system. The electrophysiological properties of the rbE-II current are consistent with a type of low voltage-activated calcium channel that requires membrane hyperpolarization for maximal activity, which suggests that rbE-II may be involved in the modulation of firing patterns.

Inositol trisphosphate and calcium signalling.

Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

Physiopathology of neuronal voltage-operated calcium channels.

Voltage-operated calcium channels are multimeric transmembrane proteins crucially involved in control of calcium homeostasis. Multiple types of voltage-operated calcium channels have been described in both the nervous system and peripheral tissues. Different channels can be classified according to either their biophysical properties or their pharmacology, biochemical and molecular structure, and localization and functional role. Concentrating on neuronal cells, this paper reviews the different properties of low- and high-voltage activated channels, as well as various attempts to subdivide high-voltage activated channels into different subtypes (L, N, omega, P, etc.). The availability of selective drugs (such as dihydropyridines) and natural toxins (such as omega-Conotoxin, omega-agatoxin, and funnel-web spider toxins), which bind to specific channel subtypes, has greatly helped in channel classification. The emerging view is that there are many members of the family of voltage-operated calcium channels, each with its own molecular structure, a different pharmacology, a different localization, and possibly a different physiological role. Different calcium subtypes are selectively affected in human and animal diseases. The use of omega-Conotoxin has led to identification of the channel subtype (omega) specifically affected in Lambert-Eaton myasthenic syndrome (a human disease of neurotransmission), and has permitted development of new diagnostic approaches to the disease.

Rat brain expresses a heterogeneous family of calcium channels.

We describe the isolation and characterization of several rat brain cDNAs that are homologous to the alpha 1 subunit of heart and skeletal muscle dihydropyridine-sensitive Ca channels. Northern blot analysis of 32 cDNAs shows that they can be grouped into four distinct classes (A, B, C, and D), each corresponding to a distinct hybridization pattern of brain mRNAs. Southern blot and DNA sequencing suggest that each class of cDNA represents a distinct gene or gene family. In the regions sequenced, the rat brain class C and D gene products share approximately 75% amino acid identity with the rabbit skeletal muscle Ca channel. In addition, the class C polypeptide is almost identical to the rabbit cardiac Ca channel (97% identity). In contrast, the rat brain class A and B cDNAs are more distantly related to dihydropyridine-sensitive Ca channels (47-64% amino acid identity) and to the brain class C and D genes (51-55% amino acid identity). To examine the functional significance of the isolated brain cDNAs, hybrid depletion experiments were performed in Xenopus oocytes. Antisense oligonucleotides against class A and B cDNAs each partially inhibited, and a class C oligonucleotide almost fully inhibited, the expression of Ba current in rat brain mRNA injected oocytes; but none of the oligonucleotides affected the expression of voltage-gated Na or K conductances. The clone characterization and sequencing results demonstrate that a number of distinct, yet related, voltage-gated Ca-channel genes are expressed in the brain. The antisense oligonucleotide experiments specifically show that one or several of the Ca-channel classes are related to the Ca channels observed in rat brain mRNA injected oocytes.

Calcium Channels

This chapter discusses the different types and tools to characterize calcium channels. It focuses on the voltage-dependent calcium channels in plasma membranes from mammalian electrically excitable cells. The calcium channel drugs are treated as structural and functional tools. The chapter also presents some electrophysiological studies of calcium channels. Calcium channels are among the Methuselahs of ion channels. The diversity and the complexity of the physiological control and pharmacological modulation of the calcium channel family have become apparent. The plasma membrane of excitable cells contains several types of calcium channels. They are distinguished by voltage dependence, unitary conductance, selectivity, and by pharmacology. In many cells, multiple channel types are observed, but there are also some membranes where only one type appears to exist. The different types (termed L, T, and N) are well characterized, especially in heart and neuronal tissue. The L type is predominant in heart and smooth muscle cells; the L, T and N types are found in nerve cells. The calcium channels can be characterized biochemically only by specificity of drug binding.