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.

Voltage-gated ion channels contain a voltage-sensing domain and a pore domain. Two families of proteins have been identified that contain the voltage-sensing domain but no pore domain, the family of voltage-sensitive phosphatases and the family of voltage-gated proton channels. Hv1, the only known voltage-gated proton channel, has clinical relevance, having been found at high expression levels in cancer cell lines, and the inhibition of Hv1 has shown to slow the growth of cancer cells. Previously, there were no known blockers of this channel that could be developed for therapeutic use. Here I have identified two intracellular blockers of Hv1. The first, 2GBI, is an open channel blocker accessible from the intracellular side. The second, ClGBI, is also an open channel blocker but accessible from the extracellular side. Through mutagenesis of the Hv1 channel and electrophysiology techniques, we have investigated the binding environment of this inhibitor and studied the mechanism of intracellular gating in Hv1. Next, due to the clinical significance of Hv1, we searched for related voltage-sensitive proteins without a pore domain, and identified Hv1's closest related protein, called HVRP1. Using a combination of techniques including in situ hybridization, immunocytochemistry, and electrophysiology, I have begun initial characterization of this new member of the family of voltage-sensitive proteins without a pore domain. The novelty of this research represents an entirely new family of proteins whose function is yet unknown, but likely to be a function yet unassociated with pore-domain-lacking voltage-sensitive proteins.

Microbial infections of the cornea (keratitis) cause an estimated 1 million cases of visual impairment and blindness worldwide each year. Treatment includes antimicrobial agents followed by corticosteroids to reduce inflammation and inhibit fibrosis. However, corticosteroids have undesirable side effects including increased intraocular pressure and risk for glaucoma. The Hv1 voltage-gated proton channel functions in the regulation of pH and NOX-mediated ROS production and has been implicated in cancer, ischemic stroke, CNS demyelination, traumatic brain injury and spinal cord injury and presents a target for therapeutic intervention. However, the role of this proton channel in ocular disease has not been examined. The immune response to invading microorganisms is characterized by recruitment of neutrophils to the site of infection which produce reactive oxygen species (ROS). Nevertheless, if produced in excess or prolonged periods of time, ROS can cause tissue damage. Using a recently discovered potent inhibitor of the Hv1 proton channel called Hv1 Inhibitor Flexible (HIF), we show that in PMA-, curdlan-, zymosan- and ΔpscD-stimulated murine bone marrow and human neutrophils, HIF inhibits ROS production in a dose-dependent manner. Our data also show that Hvcn1-/- mice, which lack the Hv1 voltage-gated proton channel, develop more severe corneal disease. However, we found that there was no difference in neutrophils and monocytes infiltration into the corneas, indicating that the inability of Hvcn1-/- mice to eliminate the bacteria is not due to a defect in cell migration and infiltration. Our results suggest that HIF targeting of the Hv1 voltage-gated proton channel together with antibiotics has a strong potential for treatment of microbial keratitis.