Abstract

Somatic mutations are common in cancer, with only a few driving the progression of the disease, while most are silent passengers. Some mutations may hinder or even reverse cancer progression. The voltage-gated proton channel (HV1) plays a key role in cellular pH homeostasis and shows increased expression in several malignancies. Inhibiting HV1 in cancer cells reduces invasion, migration, proton extrusion, and pH recovery, impacting tumor progression. Focusing on HVCN1, the gene coding for the human voltage-gated proton channel (hHV1), 197 mutations were identified from three databases: 134 missense mutations, 51 sense mutations, and 12 introducing stop codons. These mutations cluster in two hotspots: the central region of the N-terminus and the region coding for the S4 transmembrane domain, which contains the channel’s voltage sensor. Five somatic mutations within the S4 segment (R205W, R208W, R208Q, G215E, and G215R) were selected for electrophysiological analysis and MD simulations. The findings reveal that while all mutants remain proton-selective, they all exhibit reduced effective charge displacement and proton conduction. The mutations differentially affect hHV1 kinetics, with the most pronounced effects observed in the two Arg-to-Trp substitutions. Mutation of the first voltage-sensing arginine (R1) to tryptophan (R205W) causes proton leakage in the closed state, accelerates channel activation, and diminishes the voltage dependence of gating. Except for R205W, the mutations promote the deactivated channel configuration. Altogether, these data are consistent with impairment of hHV1 function by mutations in the S4 transmembrane segment, potentially affecting pH homeostasis of tumor cells.