Abstract
The voltage gated proton channel Hv1 has a remarkable architecture. It contains only a voltage sensing domain (VSD, homologous to VSDs of classic tetrameric voltage gated cation channels like Shaker), but lacks a traditional pore domain. Recent studies identified key residues that determine selectivity and located crucial parts of the pore in the VSD. Hv1 is gated by voltage and also by the transmembrane pH gradient. While the fourth transmembrane segment (S4) has been shown to undergo a voltage-dependent motion, suggesting a role in voltage sensing, as in classical VSDs, the protein motions associated with pH sensing are not understood. Here, we use the technique of Patch Clamp Fluorometry to investigate conformational changes of ciona Hv1 in response to changes of internal pH. We labeled the channel at an external site of S4 with the environmentally sensitive fluorophore 2-((5(6)-Tetramethyl-rhodamine)carboxylamino)ethyl Methanethiosulfonate (MTS-TAMRA) and compared the fluorescence signal at different internal pHs and voltages in real-time. Changing internal pH can induce a conformational change visible at S4 in the resting state, without opening of the channel. This indicates that pH gradient sensing does not require a current flux through the channel's pore.
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