Engineering Hv1 Channel Mutations and Targeted Drug for Treatment of Hv1 Mutation Associated with Cancer

The voltage-gated proton channel Hv1 is crucial for maintaining cellular pH homeostasis by regulating proton movement across membranes, playing a vital role in acid extrusion and intracellular pH regulation. Dysregulation of Hv1 is associated with various diseases, including cancer, where it helps maintain an acidic tumor microenvironment that promotes tumor progression, metastasis, and apoptosis resistance. Additionally, genetic mutations in Hv1 have been linked to several diseases, with growing evidence suggesting a strong association between Hv1 mutations and cancer. However, the mechanisms by which mutations influence Hv1 function remain poorly understood, and whether these mutations can be therapeutically targeted is still unclear. This project aims to investigate the functional impact of Hv1 disease mutations and develop targeted therapeutic strategies. The research is structured into three specific aims including engineering Hv1 mutations, functional characterization of mutations on Hv1 channel, and the development of targeted drugs for Hv1 mutation. The first aim involves studying the impact of disease mutations on Hv1 proton conductance, using a novel method to overcome challenges in comparing mutated and wild-type channels, such as differential expression and protein targeting issues. The second aim employs the patch-clamp technique to investigate the effects of mutations on Hv1 channel gating and its implications for disease progression. Finally, in the third aim, we focus on the development of targeted therapeutics, utilizing 2GBI, a known Hv1 proton channel blocker. Given that most Hv1 disease mutations are located in the core of the channel, often near or at the 2GBI binding site, we engineered new 2GBI derivatives to enhance their affinity for specific mutations, such as F150C. Our results show that certain modifications increase the affinity of blockers for mutated Hv1 channels compared to the wild-type. In summary, this work offers valuable insights into the mechanisms of Hv1 mutation-related diseases and proposes novel therapeutic strategies aimed at targeting mutant Hv1 channels.