Significance: H _v 1 proton channels, since their open probability increases with depolarization and low pH, are fundamental in sustaining the suitable pH gradient for cell survival. Here, we have characterized the gating current elicited by the monomeric mutant proton channel N264R with the aim of understanding the voltage-dependent processes that control channel opening. Gating currents precede ion currents, indicating that a large fraction of gating charge is displaced before H _v 1 opening. The voltage sensor displacements are complex and consist of numerous well-defined states. However, most of the charge is displaced in a single transition that probably leads to channel opening. The positively charged arginine in the N264R channel promotes gating charge trapping in addition to blocking the proton currents.

Significance: Acid extrusion in various types of cells and the reactive oxygen species production by the NADPH oxidase in phagocytes and microglia are mediated by the voltage-gated proton channel (H _v 1). One of the biophysical properties essential for the H _v 1 function is its ΔpH-dependent gating. Here, we measured the H _v 1 gating currents’ pH dependence in the monomeric nonconducting D160N Ciona intestinalis H _v 1 mutant. We demonstrated that H _v 1 ΔpH-dependent gating is a consequence of voltage sensor modulation by direct gating currents recordings. Around 60% of the energy stored in the ΔpH is coupled to the H _v 1 voltage sensor activation. Therefore, the proton chemical energy is transduced to the voltage sensor activation in H _v 1 to produce ΔpH-dependent gating.

Aquaporins (AQPs) are small transmembrane tetrameric proteins that facilitate water, solute and gas exchange. Their presence has been extensively reported in the biological membranes of almost all living organisms. Although their discovery is much more recent than ion transport systems, different biophysical approaches have contributed to confirm that permeation through each monomer is consistent with closed and open states, introducing the term gating mechanism into the field. The study of AQPs in their native membrane or overexpressed in heterologous systems have experimentally demonstrated that water membrane permeability can be reversibly modified in response to specific modulators. For some regulation mechanisms, such as pH changes, evidence for gating is also supported by high-resolution structures of the water channel in different configurations as well as molecular dynamics simulation. Both experimental and simulation approaches sustain that the rearrangement of conserved residues contributes to occlude the cavity of the channel restricting water permeation. Interestingly, specific charged and conserved residues are present in the environment of the pore and, thus, the tetrameric structure can be subjected to alter the positions of these charges to sustain gating. Thus, is it possible to explore whether the displacement of these charges (gating current) leads to conformational changes? To our knowledge, this question has not yet been addressed at all. In this review, we intend to analyze the suitability of this proposal for the first time.

Significance: Immunosuppression by myeloid-derived suppressor cells (MDSC), especially near tumor surfaces, involves the extracellular production of reactive oxygen species (ROS). ROS generation in MDSC occurs during the oxidation of NADPH to NADP+, which NOX2 catalyzes. ROS react with the T cell receptor complex, abolishing the antigen presentation, which blocks the immune system elimination of the tumor cells. Extrusion of protons from MDSC by voltage-gated proton channel (H_v1) sustains ROS production. Here, we demonstrate the expression of H_v1 in mouse MDSC. In this way, H_v1 present in MDSC becomes a potential cancer therapeutic target since its inhibition seems to diminish immunosuppression activity in the tumoral microenvironment, allowing cancer cells to be attacked by the immune system.

The accessory β1 subunit modulates the Ca²⁺- and voltage-activated K⁺ (BK) channel gating properties mainly by increasing its apparent Ca²⁺ sensitivity. β1 plays an important role in the modulation of arterial tone and blood pressure by vascular smooth muscle cells (SMCs). 17β-estradiol (E2) increases the BK channel open probability (P_o) in SMCs, through a β1 subunit-dependent modulatory effect. Here, using molecular modeling, bioinformatics, mutagenesis, and electrophysiology, we identify a cluster of hydrophobic residues in the second transmembrane domain of the β1 subunit, including the residues W163 and F166, as the binding site for E2. We further show that the increase in P_o induced by E2 is associated with a stabilization of the voltage sensor in its active configuration and an increase in the coupling between the voltage sensor activation and pore opening. Since β1 is a key molecular player in vasoregulation, the findings reported here are of importance in the design of novel drugs able to modulate BK channels.

Arachidonic acid (AA), a compound secreted by Sertoli cells (SC) in a FSH-dependent manner, is able to induce the release of Ca²⁺ from internal stores in round spermatids and pachytene spermatocytes. In this study, the possible site(s) of action of AA in round spermatids, the signalling pathways associated and the intracellular Ca²⁺ stores targeted by AA-induced signalling were pharmacologically characterized by measuring intracellular Ca²⁺ using fluorescent Ca²⁺ probes. Our results suggest that AA acts by interacting with a fatty acid G protein coupled receptor, initiating a G protein signalling cascade that may involve PLA2 and ERK activation, which in turn opens intracellular ryanodine-sensitive channels as well as NAADP-sensitive channels in acidic intracellular Ca²⁺ stores. The results presented here also suggest that AMPK and PKA modulate this AA-induced Ca²⁺ release from intracellular Ca²⁺ stores in round spermatids. We propose that unsaturated free fatty acid lipid signalling in the seminiferous tubule is a novel regulatory component of rat spermatogenesis.