Activation of the intrinsic aqueous water-wire proton conductance (GAQ) in Hv1 channels is controlled by changes in membrane potential and the transmembrane pH gradient (ΔpH). The mechanism by which changes in ΔpH affect the apparent voltage dependence of GAQ activation is not understood. In order to measure voltage sensor (VS) activation in Hv1, we mutated a conserved Arg residue in the fourth helical segment (S4) to His and measured H+ currents under whole-cell voltage clamp in transfected HEK-293 cells. Consistent with previous studies in VS domain containing proteins, we find that Hv1 R205H mediates a robust resting-state H+ ‘shuttle’ conductance (GSH) at negative membrane potentials. Voltage-dependent GSH gating is measured at more negative voltages than the activation GAQ, indicating that VS activation is thermodynamically distinct from opening of the intrinsic H+ permeation pathway. A hallmark biophysical feature of Hv1 channels is a ~-40 mV/pH unit shift in the apparent voltage dependence of GAQ gating. We show here that changes pHO are sufficient to cause similar shifts in GSH gating, indicating that GAQ inherits its pH dependence from an early step in the Hv1 activation pathway. Furthermore, we show for the first time that Hv1 channels manifest a form of electromechanical coupling VS activation and GAQ pore opening. Second-site mutations of D185 markedly alter GAQ gating without affecting GSH gating, indicating that D185 is required for a late step in the activation pathway that controls opening of the aqueous H+ permeation pathway. In summary, this work demonstrates that the Hv1 activation pathway contains multiple transitions with distinct voltage and pH dependencies that have not been previously identified. The results reported here novel insight into the mechanism of VS activation in Hv1 and raise fundamental questions about the nature of pH-dependent gating and electromechanical coupling in related VS domain-containing ion channels and phosphatases.

It is clear that the voltage-gated proton channel HVCN1 plays an essential role in a range of cell types, in particular immune cells. Previous published work has confirmed the existence of proton channels in both murine and human macrophages. However, the role of HVCN1 in macrophages has not been investigated. Given that the current literature on voltage-gated proton channels in immune cells has found HVCN1 to be involved in several cellular processes (such as the respiratory burst and signalling events) it is important to establish its functional role in macrophages, which are a crucial constituent of the immune system. The aim of my thesis was to investigate the function of voltage-gated proton channels in macrophages with the use of mice with a disrupting mutation within the Hvcn1 gene, which results in HVCN1 loss. In particular, I wanted to address how Hvcn1-/- macrophages responded to LPS activation. I hypothesised that HVCN1 regulates the respiratory burst of macrophages and that it potentially modulates mitochondrial ROS production, and in doing so, may affect several functional aspects of macrophage biology.