Coupling of voltage- and pH-sensing in the Hv1 proton channel

• Applicant: Dr. Thomas K. Berger
• Subject Area: Biophysics
• Term: from 2015 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 282132635

The proton channel Hv1 belongs to the superfamily of voltage-gated cation channels and is expressed in a variety of cell types. Hv1 controls acid secretion in lung epithelial cells, pH regulation and maturation in human sperm, histamine release in basophils, and the innate immune response of macrophages like microglia. The channel structure is unique: Hv1 contains only a voltage-sensing domain but lacks a traditional pore domain. I identified two key residues in transmembrane segments S1 and S4 that determine the cation selectivity of Hv1 (Berger & Isacoff, Neuron 2011). Channel opening appears to happen concomitantly with a dynamic and unique assembly of the selectivity filter. More recently, I could identify a specific role of S1 in channel gating (Mony*, Berger* & Isacoff, Nat Struct Mol Biol 2015). The voltage dependence of Hv1 is set by the transmembrane pH gradient. Asymmetric pH conditions, e.g. an acidification of the intracellular side, leads to a leftward shift of the channel's activation curve by approximately -40 mV per pH unit. This pH dependence determines the channel's physiological role. The molecular basis of this mystical pH-gradient sensing is unknown and the question of the research proposal. In project 1, I will determine which channel states and transitions are pH-dependent. Hv1 transitions from the closed to the open state by at least two conformational changes. I will investigate which transitions depend not only on voltage but also on the pH gradient across the membrane. Conformational changes will be detected with the patch-clamp fluorometry (PCF) technique (Berger & Isacoff Meth Mol Biol 2015) and the substituted cysteine accessibility method. I already established both techniques for Hv1. In addition, I will identify conformational changes with fluorescent unnatural amino acids. In Project 2, I plan to identify the pH sensor of Hv1. Crucial for its identification is to understand the coupling of voltage- and pH-sensing in Hv1. I will alter Hv1 so that pH-sensing is abolished but voltage-sensing preserved. To this end, I will introduce single or multiple mutations in Hv1 and create chimeras of Hv1 and parts of other ion channels. Taken together, central questions about structure-function relationships, pH-gradient sensing and the channel gating process of Hv1 will be addressed using state-of-the-art electrophysiological, fluorometric and molecular techniques. The insights will advance our knowledge on Hv1 and on voltage-sensing domains in general.

• DFG Programme: Research Grants