Juan Carlos Gomora

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.

Functional expression of voltage-gated sodium channels in primary cultures of human cervical cancer

Cervical cancer (CaC) is the third most frequent cause of death from cancer among women in the world and the first in females of developing countries. Several ion channels are upregulated in cancer, actually potassium channels have been suggested as tumor markers and therapeutic targets for CaC. Voltage‐gated sodium channels (VGSC) activity is involved in proliferation, motility, and invasion of prostate and breast cancer cells; however, the participation of this type of channels in CaC has not been explored. In the present study, we identified both at the molecular and electrophysiological level VGSC in primary cultures from human cervical carcinoma biopsies. With the whole cell patch clamp technique, we isolated and identified a voltage‐gated Na+ current as the main component of the inward current in all investigated cells. Sodium current was characterized by its kinetics, voltage dependence, sensitivity to tetrodotoxin (TTX) block and dependence to [Na+]o. By analyzing the expression of mRNAs encoding TTX‐sensitive Na+ channel α subunits with standard RT‐PCR and specific primers, we detected Nav1.2, Nav1.4, Nav1.6, and Nav1.7 transcripts in total RNA obtained from primary cultures and biopsies of CaC. Restriction enzyme analysis of PCR products was consistent with the molecular nature of the corresponding genes. Notably, only transcripts for Nav1.4 sodium channels were detected in biopsies from normal cervix. The results show for the first time the functional expression of VGSC in primary cultures from human CaC, and suggest that these channels might be considered as potential molecular markers for this type of cancer. J. Cell. Physiol. 210: 469–478, 2007.

Alternative splicing of the rat Cav3.3 T-type calcium channel gene produces variants with distinct functional properties1

Molecular diversity in T‐type Ca2+ channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Cav3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage‐dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter‐related, suggesting an interaction. We conclude that alternative splicing of the Cav3.3 gene produces channels with distinct properties.

Overexpression of NaV1.6 channels is associated with the invasion capacity of human cervical cancer

Functional activity of voltage‐gated sodium channels (VGSC) has been associated to the invasion and metastasis behaviors of prostate, breast and some other types of cancer. We previously reported the functional expression of VGSC in primary cultures and biopsies derived from cervical cancer (CaC). Here, we investigate the relative expression levels of VGSC subunits and its possible role in CaC. Quantitative real‐time PCR revealed that mRNA levels of NaV1.6 α‐subunit in CaC samples were ∼40‐fold higher than in noncancerous cervical (NCC) biopsies. A NaV1.7 α‐subunit variant also showed increased mRNA levels in CaC (∼20‐fold). All four NaVβ subunits were also detected in CaC samples, being NaVβ1 the most abundant. Proteins of NaV1.6 and NaV1.7 α‐subunits were immunolocalized in both NCC and CaC biopsies and in CaC primary cultures as well; however, although in NCC sections proteins were mainly relegated to the plasma membrane, in CaC biopsies and primary cultures the respective signal was stronger and widely distributed in both cytoplasm and plasma membrane. Functional activity of NaV1.6 channels in the plasma membrane of CaC cells was confirmed by whole‐cell patch‐clamp experiments using Cn2, a NaV1.6‐specific toxin, which blocked ∼30% of the total sodium current. Blocking of sodium channels VGSC with tetrodotoxin and Cn2 did not affect proliferation neither migration, but reduced by ∼20% the invasiveness of CaC primary culture cells in vitro assays. We conclude that NaV1.6 is upregulated in CaC and could serve as a novel molecular marker for the metastatic behavior of this carcinoma.

Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca2+ channel α2δ-1 auxiliary subunit

Voltage-gated calcium (Ca(V)) channels are transmembrane proteins that form Ca(2+)-selective pores gated by depolarization and are essential regulators of the intracellular Ca(2+) concentration. By providing a pathway for rapid Ca(2+) influx, Ca(V) channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. Ca(V) channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α(1) subunit, which typically contains associated β and α(2)δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α(2)δ-1 is post-translationally cleaved into an extracellular α(2) polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α(2)δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α(2)) and Cys1047 (in the extracellular domain of δ) prevented the association of the α(2) and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α(2)δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (Ca(V)2.2α(1)/β(3)) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α(2)δ-1 function.

Niflumic acid blocks native and recombinant T-type channels.

Voltage‐dependent calcium channels are widely distributed in animal cells, including spermatozoa. Calcium is fundamental in many sperm functions such as: motility, capacitation, and the acrosome reaction (AR), all essential for fertilization. Pharmacological evidence has suggested T‐type calcium channels participate in the AR. Niflumic acid (NA), a non‐steroidal anti‐inflammatory drug commonly used as chloride channel blocker, blocks T‐currents in mouse spermatogenic cells and Cl− channels in testicular sperm. Here we examine the mechanism of NA blockade and explore if it can be used to separate the contribution of different CaV3 members previously detected in these cells. Electrophysiological patch‐clamp recordings were performed in isolated mouse spermatogenic cells and in HEK cells heterologously expressing CaV3 channels. NA blocks mouse spermatogenic cell T‐type currents with an IC50 of 73.5 µM, without major voltage‐dependent effects. The NA blockade is more potent in the open and in the inactivated state than in the closed state of the T‐type channels. Interestingly, we found that heterologously expressed CaV3.1 and CaV3.3 channels were more sensitive to NA than CaV3.2 channels, and this drug substantially slowed the recovery from inactivation of the three isoforms. Molecular docking modeling of drug‐channel binding predicts that NA binds preferentially to the extracellular face of CaV3.1 channels. The biophysical characteristics of mouse spermatogenic cell T‐type currents more closely resemble those from heterologously expressed CaV3.1 channels, including their sensitivity to NA. As CaV3.1 null mice maintain their spermatogenic cell T‐currents, it is likely that a novel CaV3.2 isoform is responsible for them. J. Cell. Physiol. 227: 2542–2555, 2012.

Insulin-mediated upregulation of T-type Ca2+ currents in GH3 cells is mediated by increased endosomal recycling and incorporation of surface membrane Cav3.1 channels.

Growth factors and hormones have both short- and long-term regulatory effects on the functional expression of voltage gated Ca2+ (CaV) channels. In particular, it has been reported that chronic treatment with insulin upregulates T-type channel membrane expression, leading to an increase in current density in clonal pituitary GH3 cells. Though this regulatory action may result from alterations in gene expression, recent studies have demonstrated also that endosomal trafficking provides a mechanism for dynamic changes in CaV channel membrane density. Therefore, in the present work we sought to determine whether the actions of insulin on T-type channel functional expression are mediated by transcriptional and/or post-transcriptional mechanisms. Using real-time RT-PCR and semi-quantitative western blot we found no changes after treatment in the transcript and protein levels of Cav3.1, the T-type channel isoform preferentially expressed in the GH3 cells. Consistent with this, transcriptional studies using a luciferase reporter assay suggested that insulin treatment does not affect the Cav3.1 promoter activity. In contrast, patch-clamp recordings on HEK-293 cells stably expressing Cav3.1 channels showed a significant increase in current density after treatment, suggesting that the effects of insulin may require post-transcriptional regulation. In line with this, disruption of the endosomal recycling pathway using Brefeldin A and a dominant negative mutant of the small GTPase Rab11a prevented the stimulatory effects of insulin on Cav3.1 channels in HEK-293 cells. These results may help explain the effects of insulin on T-type channels and contribute to our understanding of how endosomal recycling impacts the functional expression of CaV channels.

Block of Human CaV3 Channels by the Diuretic Amiloride

Previous studies in native T-type currents have suggested the existence of distinct isoforms with dissimilar pharmacology. Amiloride was the first organic blocker to selectively block the native T-type calcium channel, but the potency and mechanism of block of this drug on the three recombinant T-type calcium channels (CaV3.1, CaV3.2, and CaV3.3) have not been systematically determined. The aim of the present study was to investigate whether there is differential block of CaV3 channels by amiloride, to establish the mechanism of block, and to obtain insights into the amiloride putative binding sites in CaV3 channels. By performing whole-cell patch-clamp recordings of human embryonic kidney 293 cells stably expressing human CaV3 channels, we found that amiloride blocked the human CaV3 channels in a concentration-response manner; the IC50 for CaV3.2 channels (62 μM) was 13-fold lower than that for CaV3.1 and CaV3.3. Block is voltage-independent (except for CaV3.3 channels) and targets mainly closed-state channels, although a small use-dependent component was observed in CaV3.1 channels. In addition, amiloride block of CaV3.2 channels is mainly due to an extracellular effect, whereas in CaV3.1 and CaV3.3 channels, the amiloride inhibition is equally effective from both sides of the membrane. The results demonstrate that amiloride blocks human CaV3 channels differentially through a mechanism involving mainly the closed state of the channel and suggest a negative allosteric interaction with at least two putative binding sites with different affinities. The preferential block of CaV3.2 channels labels amiloride as the only organic blocker to be selective for any T-type channel.

Postnatal decrease of sodium current density in rat pituitary melanotropes following the onset of dopaminergic innervation

Peptide secretion from rat melanotropes is tonically inhibited by a dopaminergic synaptic input that develops after birth and acts through D2 dopamine receptors. In this study, whole-cell Na(+) currents were recorded from melanotropes that were isolated from rat pituitary intermediate lobes at postnatal days 1-20 (P1-P20) and maintained in culture for 5-24 h. Coincident with the development of innervation, melanotropes exhibited a progressive decrease in peak Na(+) current density from P3 to P14. The decrease involved a 50% reduction in maximal Na(+) conductance with no detectable changes in channel gating. Subcutaneous injections of the D2 antagonist sulpiride, applied from P11 to P13, restored melanotrope Na(+) channel activity to pre-innervation levels. Thus, the activation of D2 receptors by the dopaminergic input reduces the functional expression of Na(+) channels in melanotropes.

CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10−6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5–10, p = 3.3 x 10−6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.