Background: Spider venoms contain peptide toxins that modify ion channel currents, primarily in excitable insect cells. Research has revealed various peptides with pharmacological properties affecting mammalian species, including immune cell ion channels like the voltage-gated proton channel Hv1. This channel facilitates reactive oxygen species (ROS) production during the respiratory burst, contributing to inflammation and carcinogenesis by maintaining an acidic tumor microenvironment that supports tumor growth, invasion, and resistance to apoptosis. Experimental Approach: In this study, six animal venoms underwent screening against hHv1 using the patch clamp technique. The complete venom extracted from the spider Grammostola rosea resulted in the modulation of hHv1 expressed in CHO cells during whole-cell patch clamp measurements. HPLC reverse-phase fractionation of the venom showed that two peptide fractions #7 (GsAF-I) and #9 (GsAF-II) were active . Key Results: GsAF-I and GsAF-II inhibited Hv1 with micromolar Kd values in a membrane potential-dependent manner. Voltage ramp protocol revealed a shift in the Hv1 activation threshold toward a more positive potential. Voltage step protocol showed slowed activation kinetics and accelerated deactivation kinetics, suggesting stabilization of the closed state. The effects of both peptides were reversible. The membrane potential dependence of the inhibition was uniquely analysed and presented in this study. Conclusion and Implications: This is the first study to investigate in detail the inhibitory effect of peptides from animal venoms on the human Hv1 ion channel. The found two peptides effectively modify proton currents, highlighting their potential as lead compounds for drug development.

not-yet-known not-yet-known not-yet-known unknown The voltage-gated proton channel (Hv1) selectively transports protons (H⁺) across biological membranes in response to membrane potential changes. Hv1 assembles 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. Hv1 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. Hv1 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, Hv1 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 Hv1 can be divided into two broad groups: inhibitors and activators. Hv1 inhibitors can be simple ions, small molecules, lipids, and peptides. In contrast, fewer Hv1 activators are known, including Albumin, NH29, Quercetin, and arachidonic acid. The mechanism of action of some inhibitors is well described, but not all. Hv1 modulation has profound effects on cellular physiology, especially under stress or pathological conditions, like cancer and inflammation. The therapeutic application of selective Hv1 inhibitors or activators could be a very promising strategy in the treatment of several serious diseases.