UC Irvine

Voltage-gated proton channels (Hvs) are membrane proteins that belong to the superfamily of voltage-gated ion channels (VGICs). They are dimers consisting of two voltage sensing domains (VSDs), which resemble the ones from other VGICs, but lack the pore domain (PD) commonly found in other VGICs. They regulate cellular pH homeostasis and their activity is frequently coupled with reactive oxygen species (ROS) production by NADPH oxidase (NOX) enzymes, a critical process in the elimination of pathogens by phagocytes through the respiratory burst.The structural determinants that set the voltage range of activation of Hv channels are poorly understood, and so is the mechanism underlying the dependence on co-stimuli, such as intracellular acidification and membrane stretch. Here, I exploited the functional diversity of Hv channels from distantly related organisms to identify protein regions responsible for the modulation of channel activation and to uncover new mechanisms of co-stimulation. By comparing Hv homologs from different species of fungi, I found that the distinctively negative voltage range of activation of some fungal Hv channels is controlled by their extracellular peripheral regions. By comparing Hv homologs from different species of plants, I identified Hv channels that require mechanical priming prior to voltage-dependent activation, a property most likely controlled by their S4 transmembrane segment, which is divergent between angiosperm and gymnosperm plants. My findings suggest that evolution has tuned the biophysical properties of these channels to match the distinct physiological contexts in which they operate. In human and other animals, the Hv1 channel is widely expressed in the immune system, including in B and T lymphocytes, macrophages, neutrophils, and basophils as well as in the microglia within the central nervous system (CNS). Previous studies have found that Hv1 activity plays a role in the progression of various types of cancers (e.g., B cell lymphoma, breast cancer, colorectal cancer), and impairs the recovery from CNS damage caused by ischemic stroke, traumatic brain injury, and spinal cord injury. Therefore, developing inhibitors targeting Hv1 could provide effective treatments for a variety of pathological conditions. Based on our understanding of how a previous generation of guanidine mimics (e.g., 2GBI and ClGBI) inhibit Hv1, we rationally designed the next generation of compounds, named HIFs (Hv1 Inhibitor Flexible). I found that some HIF molecules inhibit Hv1 at lower concentrations compared to 2GBI, and possess desirable features for further drug development. I characterized the mechanism of HIF-mediated inhibition and found evidence of two distinct binding sites: one located deep into the intracellular vestibule of the channel and shared with 2GBI, and one located in a shallower part of the vestibule, closer to the inner mouth of the channel. The existence of the second binding site could explain some of the desirable pharmacological properties that distinguish HIFs form first-generation inhibitors.