Voltage-gated proton (HV) channels have been found in many mammalian cell types, including blood cells, lung epithelia, skeletal muscle, and microglia. Of particular importance, HV channels have been shown to play a crucial role in blood cells: HV channels in macrophages are essential for the generation of reactive oxygen species during the respiratory burst, which is critical to the process of phagocytosis and the destruction of foreign pathogens. Our long-term goal is to understand the molecular mechanism of HV channel function in macrophages and other cell types. Recently, the molecular identify of HV channels was discovered. HV was found to be homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels. Kv channels are tetrameric channels with 6 transmembrane (TM) segments and a pore domain. However, HV channels have only the first 4 TMs and lack a typical pore domain. We recently showed that HV channels are dimers, with each subunit having its own proton pathway. Many questions about their molecular function remain unanswered. How do two HV subunits come together to form a dimeric HV channel? How does voltage activate HV channels and how does macrophage activation alter the activity of HV channels? What constitutes the proton permeation pathway in HV channels? The research objectives of this grant are to determine the mechanism of dimerization of HV subunits, to determine how dimerization affects the activity and cooperativity of HV subunits, and to identify residues lining the proton conduction pathway in HV channels. The results of the proposed work will provide a greater understanding of the structure of the voltage-gated proton channel HV and will provide a first step in understanding how the activity of this protein is regulated. The proposed work is highly significant to understanding the basis of innate immunity as well as the pathology underlying asthma. HV is a crucial component in the function of phagocytes of the immune system, and it is also an important factor in the lungs during asthma. A greater understanding of the structure and regulation of HV will therefore provide new insight into the role that this protein plays in both disease and in normal immune function, and could provide novel avenues for therapeutic intervention in a number of pathologies. Most significantly, the regulation of HV could be a potential drug target for modulating the inflammatory response of the immune system as well as for the treatment of asthma.