5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H⁺) channel (H_V1, K_d ≈ 26 μM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the K_V1.3 channel. We, therefore, tested ClGBI directly on hK_V1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hH_V1 (K_d ≈ 72 μM). We then further investigated ClGBI selectivity on the hK_V1.1, hK_V1.4-IR, hK_V1.5, hK_V10.1, hK_V11.1, hK_Ca3.1, hNa_V1.4, and hNa_V1.5 channels. Our results show that, besides H_V1 and K_V1.3, all other off-target channels were inhibited by ClGBI, with K_d values ranging from 12 to 894 μM. Based on our comprehensive data, ClGBI has to be considered a non-selective hH_V1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.

Voltage-gated K⁺ channels have distinct gates that regulate ion flux: the activation gate (A-gate) formed by the bundle crossing of the S6 transmembrane helices and the slow inactivation gate in the selectivity filter. These two gates are bidirectionally coupled. If coupling involves the rearrangement of the S6 transmembrane segment, then we predict state-dependent changes in the accessibility of S6 residues from the water-filled cavity of the channel with gating. To test this, we engineered cysteines, one at a time, at S6 positions A471, L472, and P473 in a T449A Shaker-IR background and determined the accessibility of these cysteines to cysteine-modifying reagents MTSET and MTSEA applied to the cytosolic surface of inside-out patches. We found that neither reagent modified either of the cysteines in the closed or the open state of the channels. On the contrary, A471C and P473C, but not L472C, were modified by MTSEA, but not by MTSET, if applied to inactivated channels with open A-gate (OI state). Our results, combined with earlier studies reporting reduced accessibility of residues I470C and V474C in the inactivated state, strongly suggest that the coupling between the A-gate and the slow inactivation gate is mediated by rearrangements in the S6 segment. The S6 rearrangements are consistent with a rigid rod-like rotation of S6 around its longitudinal axis upon inactivation. S6 rotation and changes in its environment are concomitant events in slow inactivation of Shaker K_V channels.

The hEag1 (Kv10.1) K⁺ channel is normally found in the brain, but it is ectopically expressed in tumor cells, including osteosarcoma. Based on the pivotal role of ion channels in osteogenesis, we tested whether pharmacological modulation of hEag1 may affect osteogenic differentiation of osteosarcoma cell lines. Using molecular biology (RT-PCR), electrophysiology (patch-clamp) and pharmacology (astemizole sensitivity, IC₅₀ = 0.135 μM) we demonstrated that SaOS-2 osteosarcoma cells also express hEag1 channels. SaOS-2 cells also express to KCa1.1 K⁺ channels as shown by mRNA expression and paxilline sensitivity of the current. The inhibition of hEag1 (2 μM astemizole) or KCa1.1 (1 mM TEA) alone did not induce Ca²⁺ deposition in SaOS-2 cultures, however, these inhibitors, at identical concentrations, increased Ca²⁺ deposition evoked by the classical or pathological (inorganic phosphate, Pi) induction pathway without causing cytotoxicity, as reported by three completer assays (LDH release, MTT assay and SRB protein assay). We observed a similar effect of astemizole on Ca²⁺ deposition in MG-63 osteosarcoma cultures as well. We propose that the increase in the osteogenic stimuli-induced mineral matrix formation of osteosarcoma cell lines by inhibiting hEag1 may be a useful tool to drive terminal differentiation of osteosarcoma.

Brugada syndrome (BrS) is a genetically determined cardiac arrhythmogenic syndrome with increased risk of sudden cardiac death. BrS is mostly caused by mutations in SCN5A gene encoding the primary ɑ-subunit of the cardiac sodium channel Na _V 1.5. We aimed at characterizing the functional alterations caused by the R893C mutation, identified in a proband diagnosed with BrS, and establishing whether the mutation is associated with BrS. Although several mutations have been reported in the close vicinity of R893 , the functional role of this region remains unknown and, in addition, exploring SCN5A mutations in patients with inherited arrhythmogenic syndromes is critical for understanding the pathogenesis of arrhythmias. The mutations were introduced by site-directed mutagenesis. The variants were transiently expressed in CHO cells and potassium currents were measured using the whole-cell patch clamp technique. Patch clamp recordings have demonstrated that R893C almost completely abolished the sodium current, I _Na , though the mutation did not exert dominant-negative effect on wild-type Na _V 1.5 channels. We also observed significant decrease in channel activation and a depolarized shift of steady–state inactivation curve, however, the kinetics of inactivation and recovery from fast inactivation were not changed by the mutation. Moreover, the reducing agent Dithiotreitol partially restored the normal function of Na _V 1.5 in the R893C mutant highlighting a likely mechanism for loss of conduction via formation of disulphide bridges. We showed that R893H channels also failed to produce any detectable I _Na that confirms the importance of the highly conserved R893 in gating. Our study reveals R893C is a loss-of-function mutation with altered electrophysiological characteristics of Na _V 1.5. Thus, R893C may contribute to the BrS phenotype of the proband. Our findings may facilitate the understanding of the mechanisms of arrhythmogenesis in BrS, as it helps to identify mutational hotspots in BrS. Moreover, our work may improve novel gene therapy and new therapeutic drug design targeting Na _V 1.5 channelopathies.

The voltage-gated proton channel (H_v1) selectively transports protons (H⁺) across biological membranes in response to membrane potential changes. H_v1 is assembled as a dimer, and unlike most voltage-gated ion channels, it lacks a traditional central pore domain; instead, the voltage-sensing domain (VSD) of each monomer facilitates proton conduction via a hydrogen-bond network. H_v1 is widely expressed in various human cell types (e.g., immune cells, sperm, etc.) including tumor cells. In tumor cells, the accumulation of acidic intermediates generated by glycolysis under hypoxic conditions or ROS production leads to significant cytosolic acidification. H_v1 can remove protons from the cytosol rapidly, contributing to the adaptation of the cells to the tumor microenvironment, which may have significant consequences in tumor cell survival, proliferation, and progression. Therefore, H_v1 may be very promising not only as a tumor marker but also as a potential therapeutic target in oncology. Molecules that modulate the proton flux through H_v1 can be divided into two broad groups: inhibitors and activators. H_v1 inhibitors can be simple ions, small molecules, lipids, and peptides. In contrast, fewer H_v1 activators are known, including albumin, NH29, quercetin, and arachidonic acid. The mechanism of action of some inhibitors is well described, but not all. H_v1 modulation has profound effects on cellular physiology, especially under stress or pathological conditions, like cancer and inflammation. The therapeutic application of selective H_v1 inhibitors or activators could be a very promising strategy in the treatment of several serious diseases.