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 (Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3) have not been systematically determined. The aim of the present study was to investigate whether there is differential block of Ca(V)3 channels by amiloride, to establish the mechanism of block, and to obtain insights into the amiloride putative binding sites in Ca(V)3 channels. By performing whole-cell patch-clamp recordings of human embryonic kidney 293 cells stably expressing human Ca(V)3 channels, we found that amiloride blocked the human Ca(V)3 channels in a concentration-response manner; the IC50 for Ca(V)3.2 channels (62 µM) was 13-fold lower than that for Ca(V)3.1 and Ca(V)3.3. Block is voltage-independent (except for Ca(V)3.3 channels) and targets mainly closed-state channels, although a small use-dependent component was observed in Ca(V)3.1 channels. In addition, amiloride block of Ca(V)3.2 channels is mainly due to an extracellular effect, whereas in Ca(V)3.1 and Ca(V)3.3 channels, the amiloride inhibition is equally effective from both sides of the membrane. The results demonstrate that amiloride blocks human Ca(V)3 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 Ca(V)3.2 channels labels amiloride as the only organic blocker to be selective for any T-type channel.

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 Ca(V)3 members previously detected in these cells. Electrophysiological patch-clamp recordings were performed in isolated mouse spermatogenic cells and in HEK cells heterologously expressing Ca(V)3 channels. NA blocks mouse spermatogenic cell T-type currents with an IC(50) 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 Ca(V)3.1 and Ca(V)3.3 channels were more sensitive to NA than Ca(V)3.2 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 Ca(V)3.1 channels. The biophysical characteristics of mouse spermatogenic cell T-type currents more closely resemble those from heterologously expressed Ca(V)3.1 channels, including their sensitivity to NA. As Ca(V)3.1 null mice maintain their spermatogenic cell T-currents, it is likely that a novel Ca(V)3.2 isoform is responsible for them.

Overexpression of NaV 1.6 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 Na(V) 1.6 α-subunit in CaC samples were ∼40-fold higher than in noncancerous cervical (NCC) biopsies. A Na(V) 1.7 α-subunit variant also showed increased mRNA levels in CaC (∼20-fold). All four Na(V) ß subunits were also detected in CaC samples, being Na(V) ß1 the most abundant. Proteins of Na(V) 1.6 and Na(V) 1.7 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 Na(V) 1.6 channels in the plasma membrane of CaC cells was confirmed by whole-cell patch-clamp experiments using Cn2, a Na(V) 1.6-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 Na(V) 1.6 is upregulated in CaC and could serve as a novel molecular marker for the metastatic behavior of this carcinoma.

Characterization of the gating brake in the I-II loop of Ca(v)3.2 T-type Ca(2+) channels.

Mutations in the I-II loop of Ca(v)3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high alpha-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.

Bursting in substantia nigra pars reticulata neurons in vitro: possible relevance for Parkinson disease.

Projection neurons of the substantia nigra reticulata (SNr) convey basal ganglia (BG) processing to thalamocortical and brain stem circuits responsible for movement. Two models try to explain pathological BG performance during Parkinson disease (PD): the rate model, which posits an overexcitation of SNr neurons due to hyperactivity in the indirect pathway and hypoactivity of the direct pathway, and the oscillatory model, which explains PD as the product of pathological pattern generators disclosed by dopamine reduction. These models are, apparently, incompatible. We tested the predictions of the rate model by increasing the excitatory drive and reducing the inhibition on SNr neurons in vitro. This was done pharmacologically with bath application of glutamate agonist N-methyl-d-aspartate and GABA(A) receptor blockers, respectively. Both maneuvers induced bursting behavior in SNr neurons. Therefore synaptic changes forecasted by the rate model induce the electrical behavior predicted by the oscillatory model. In addition, we found evidence that Ca(V)3.2 Ca(2+) channels are a critical step in generating the bursting firing pattern in SNr neurons. Other ion channels involved are: hyperpolarization-activated cation channels, high-voltage-activated Ca(2+) channels, and Ca(2+)-activated K(+) channels. However, although these channels shape the temporal structure of bursting, only Ca(V)3.2 Ca(2+) channels are indispensable for the initiation of the bursting pattern.