Ricardo Felix

Calcium Channels and Ca2+ Fluctuations in Sperm Physiology

Generating new life in animals by sexual reproduction depends on adequate communication between mature and competent male and female gametes. Ion channels are instrumental in the dialogue between sperm, its environment, and the egg. The ability of sperm to swim to the egg and fertilize it is modulated by ion permeability changes induced by environmental cues and components of the egg outer layer. Ca(2+) is probably the key messenger in this information exchange. It is therefore not surprising that different Ca(2+)-permeable channels are distinctly localized in these tiny specialized cells. New approaches to measure sperm currents, intracellular Ca(2+), membrane potential, and intracellular pH with fluorescent probes, patch-clamp recordings, sequence information, and heterologous expression are revealing how sperm channels participate in fertilization. Certain sperm ion channels are turning out to be unique, making them attractive targets for contraception and for the discovery of novel signaling complexes.

Expression and differential cell distribution of low-threshold Ca2+ channels in mammalian male germ cells and sperm

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca2+ influx through voltage‐gated Ca2+ (CaV) channels. Although the electrophysiological characterization of Ca2+ currents in mature sperm has proven difficult, functional studies have revealed the presence of low‐threshold (CaV3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of CaV3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three CaV3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only CaV3.1 and CaV3.2 were detected in the head, suggesting its participation in the AR. CaV3.1 and CaV3.3 were found in the principal and the midpiece of the flagella. All CaV3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of CaV3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit CaV3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high‐threshold (HVA) CaV channels caused small but significant motility alterations. Antibodies to HVA channels detected CaV1.3 and CaV2.3 in human sperm flagella.

Intramembrane charge movement associated with endogenous K+ channel activity in HEK-293 cells.

Abstract1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable.2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl− channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl− channels to the whole-cell current.3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.

Molecular Regulation of Voltage-Gated Ca2+ Channels

Voltage-gated Ca2+ (CaV) channels are found in all excitable cells and many nonexcitable cells, in which they govern Ca2+ influx, thereby contributing to determine a host of important physiological processes including gene transcription, muscle contraction, hormone secretion, and neurotransmitter release. The past years have seen some significant advances in our understanding of the functional, pharmacological, and molecular properties of CaV channels. Molecular studies have revealed that several of these channels are oligomeric complexes consisting of an ion-conducting α1 subunit and auxiliary α2δ, β, and γ subunits. In addition, cloning of multiple CaV channel α1 subunits has offered the opportunity to investigate the regulation of these proteins at the molecular level. The regulation of CaV channels by intracellular second messengers constitutes a key mechanism for controlling Ca2 + influx. This review summarizes recent advances that have provided important clues to the underlying molecular mechanisms involved in the regulation of CaV channels by protein phosphorylation, G-protein activation, and interactions with Ca2 +-binding and SNARE proteins.

Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels.

This report describes the isolation, primary structure determination, and functional characterization of two similar toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively). KLII from P. granulatus is identical to kurtoxin from Parabuthus transvaalicus (a 63 amino-acid long toxin) whereas KLI is a new peptide containing 62 amino acid residues closely packed by four disulfide bridges with a molecular mass of 7244. Functional assays showed that both toxins, KLI and kurtoxin from P. granulatus, potently inhibit native voltage-gated T-type Ca(2+) channel activity in mouse male germ cells. In addition, KLI was shown to significantly affect the gating mechanisms of recombinant Na(+) channels and weakly block alpha(1)3.3Ca(V) channels expressed in Xenopus oocytes. KLI and kurtoxin from P. granulatus represent new probes to study the role of ion channels in germ cells, as well as in cardiac and neural tissue.

The S218L familial hemiplegic migraine mutation promotes deinhibition of Cav2.1 calcium channels during direct G-protein regulation

Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in CACNA1A, the gene encoding for the Cav2.1 subunit of voltage-gated calcium channels. Although various studies attempted to determine biophysical consequences of these mutations on channel activity, it remains unclear exactly how mutations can produce a FHM-1 phenotype. A lower activation threshold of mutated channels resulting in increased channel activity has been proposed. However, hyperactivity may also be caused by a reduction of the inhibitory pathway carried by G-protein-coupled-receptor activation. The aim of this study is to determine functional consequences of the FHM-1 S218L mutation on direct G-protein regulation of Cav2.1 channels. In HEK 293 cells, DAMGO activation of human μ-opioid receptors induced a 55% Ba2+ current inhibition through both wild-type and S218L mutant Cav2.1 channels. In contrast, this mutation considerably accelerates the kinetic of current deinhibition following channel activation by 1.7- to 2.3-fold depending on membrane potential values. Taken together, these data suggest that the S218L mutation does not affect G-protein association onto the channel in the closed state but promotes its dissociation from the activated channel, thereby decreasing the inhibitory G-protein pathway. Similar results were obtained with the R192Q FHM-1 mutation, although of lesser amplitude, which seems in line with the less severe associated clinical phenotype in patients. Functional consequences of FHM-1 mutations appear thus as the consequence of the alteration of both intrinsic biophysical properties and of the main inhibitory G-protein pathway of Cav2.1 channels. The present study furthers molecular insight in the physiopathology of FHM-1.

Down-Regulation of N-Type Voltage-Activated Ca2+ Channels by Gabapentin

Abstract1. Although the cellular and molecular mechanisms of the anticonvulsant action of gabapentin (GBP) remain incompletely described, in vitro studies have shown that GBP binds to the α2 subunit of the high voltage-activated (HVA) Ca2+ channels.2. In this report, we analyzed the effects of GBP on the functional expression of HVA Ca2+ channels in the PC12 cell line model system. Negligible inhibition of Ca2+ channel activity was observed after acute treatment, but a significant decrease in Ca2+ current amplitude was promoted by chronic exposure to GBP.3. Consistent with this, radioligand binding experiments showed a comparable reduction in the total number of membrane HVA N-type channels after GBP treatment.

Channelopathies: ion channel defects linked to heritable clinical disorders

Electrical signals are critical for the function of neurones, muscle cells, and cardiac myocytes. Proteins that regulate electrical signalling in these cells, including voltage gated ion channels, are logical sites where abnormality might lead to disease. Genetic and biophysical approaches are being used to show that several disorders result from mutations in voltage gated ion channels. Understanding gained from early studies on the pathogenesis of a group of muscle diseases that are similar in their episodic nature (periodic paralysis) showed that these disorders result from mutations in a gene encoding a voltage gated Na+ channel. Their characterisation as channelopathies has served as a paradigm for other episodic disorders. For example, migraine headache and some forms of epilepsy have been shown to result from mutations in voltage gated Ca2+channel genes, while long QT syndrome is known to result from mutations in either K+ or Na+ channel genes. This article reviews progress made in the complementary fields of molecular genetics and cellular electrophysiology which has led to a better understanding of voltage gated ion channelopathies in humans and mice.

Insights from Mouse Models of Absence Epilepsy into Ca2+ Channel Physiology and Disease Etiology

Abstract1. Changes in intracellular Ca2+ ([Ca2+]i) levels provide signals that allow neurons to respond to a host of external stimuli. A major mechanism for elevating Ca2+ ([Ca2+]i) is the influx of extracellular Ca2+ through voltage-gated channels (CaV) in the plasma membrane. in CaV due to mutations in genes encoding channel proteins are increasingly being implicated in causing disease conditions, termed channelopathies.2. Seven spontaneous mutations with cerebellar ataxia and generalized absence epilepsy have been identified in mice (tottering, leaner, rolling Nagoya, rocker, lethargic, ducky, and stargazer), and these overlapping phenotypes are directly related to mutations in genes encoding the four separate subunits that together form the multimeric neuronal CaV complex.3. The discovery and systematic analysis of these animal models is helping to clarify how different mutations affect channel function and how altered channel function produces disease.

Ghrelin inhibits proliferation and increases T-type Ca2+ channel expression in PC-3 human prostate carcinoma cells.

Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca(2+) levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca(2+) channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca(2+) channel expression.