The cytoplasmic coiled-coil mediates cooperative gating temperature sensitivity in the voltage-gated H+ channel Hv1

Abstract

Hv1/VsoP is a dimeric voltage-gated H + channel in which the gating of one subunit is reportedly coupled to that of the other subunit within the dimer. The molecular basis for dimer formation and intersubunit coupling, however, remains unknown. Here we show that the carboxy terminus ends downstream of the s4 voltage-sensor helix twist in a dimer coiled-coil architecture, which mediates cooperative gating. We also show that the temperature-dependent activation of H + current through Hv1/VsoP is regulated by thermostability of the coiled-coil domain, and that this regulation is altered by mutation of the linker between s4 and the coiled-coil. Cooperative gating within the dimer is also dependent on the linker structure, which circular dichroism spectrum analysis suggests is α-helical. our results indicate that the cytoplasmic coiled-coil strands form continuous α-helices with s4 and mediate cooperative gating to adjust the range of temperatures over which Hv1/VsoP operates.

Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel

“…The molecular mass of the coiled-coil domain protein showed a single peak corresponding to a dimer (Fig. 6A), which is consistent with our previous results from analytical ultracentrifugation (13). The signal peak was shifted in two steps toward the higher mass by adding MalPEG 10,000, where the lower mass peak corresponding to the monomer was not generated (Fig.…”

“…The thermal stability of the coiled-coil assembly was analyzed by CD spectroscopy. The temperature dependence of the CD signal showed a cooperative loss of structure, and as indicated in a previous study (13), the melting temperature (apparent T m ϭ 40.6 and 40.8°C (n ϭ 2)) was lower than those of other naturally occurring coiled-coil proteins (T m Ͼ 65°C) (Fig. 5A).…”

“…Cys-245 is located at position a of the heptad repeat, suggesting that the two sulfur atoms of Cys-245 are close to each other in the coiled-coil core. Our first structure of the coiled-coil domain shows dual conformations at Cys-245 and surrounding residues (Ser-244 and Ser-246): one involves a face-to-face formation of the Cys-245 thiols without cross-linking, and the second involves a back-to-back formation (13). This suggests that the coiled-coil structure around Cys-245 is flexible and potentially forms a disulfide bond.…”

“…VSOP/Hv1 functions as a dimer (10 -12), and the dimerization elicits a strong voltage-dependent H ϩ conductance that keenly responds to the NADPH oxidase activity (13)(14)(15). We recently identified the dimeric assembly domain of VSOP/Hv1 and solved its highresolution crystal structure (13).…”

Hydrophobic plug functions as a gate in voltage-gated proton channels

“…The transition of the closed-state monomer to the open-state monomer transports ∼1.9 e -across the membrane per subunit. This result is in agreement with the estimates of 2-to 3-e -effective gating charges per Hv1 subunit (15,38). This gating charge is mainly mediated by the motion of the charged arginines in S4 across the membrane.…”