Tombola F , Ulbrich MH , Kohout SC , Isacoff EY .

R01NS035549 NINDS NIH HHS , R01 NS035549-13 NINDS NIH HHS , R01 NS035549 NINDS NIH HHS

	Figure 2. Cooperative gating in Hv1 linked dimers made of 153C and WT subunits. (a) Cartoon representing the possible scenarios for subunit cooperativity in Hv1 linked dimers. Subunits with different colors have different activation properties. Purple shading indicates an influence of one subunit on the other. (b) Voltage dependence of proton conductances of the linked 153C–153C and WT-WT homodimers, compared to the voltage dependence of linked 153C-WT heterodimers. See Table 1 for Boltzmann fits. (c) Comparison between voltage dependence of the linked 153C-WT and WT-153C heterodimers and the prediction for the independent opening of the two pores (continuous line). pHi = pHo = 6.0. Error bars are s.e.m., n = 4–6.
	Figure 3. Cooperative gating in Hv1 dimers made of MS and ZS subunits. (a) Assembly of MTSET-sensitive (MS) and Zinc-sensitive (ZS) subunits in homo-and heterodimers. White X indicates blocked MS subunit after MTSET treatment. (b) Example of changes in normalized proton currents from membrane patches of oocytes expressing Hv1 MS (gray diamonds), Hv1 ZS (green squares), or co-expressing Hv1 MS with ZS (black circles). 25 μM Zn2+ was maintained in the extracellular solution throughout the recordings. pHi = pHo = 6.0. Black bar indicates duration of exposure to 1 mM intracellular MTSET. The RNA ratio for the co-expression of MS and ZS subunits was 3:1. (c) Quantification of inhibition produced by MTSET for the indicated conditions (black bars). From the extent of proton current inhibition the percentage of MS subunits was calculated (gray bars), as explained in the text. (d) Voltage dependence of proton conductance of Hv1 MS and Hv1 ZS, compared to the voltage dependence of the co-injection of Hv1 MS + Hv1 ZS. See Table 1 for Boltzmann fits. Each point is the average of 4–6 measurements ± s.e.m. Recording conditions and RNA ratio were the same as for panels b and c. (e) Comparison between voltage dependence of proton channels produced by co-expression of MS and ZS subunits (open circles), and voltage dependence expected in case of independent opening of the two pores (continuous line, see text).
	Figure 4. Quantification of cooperativity in Hv1. (a) Voltage dependence of activation of the MS/ZS heterodimer and the 153C-WT linked dimer fitted with the opening model shown in b. For MS/ZS Hv1: ϕMS,MS = ϕMS,ZS = ϕZS,ZS = 60 ± 35, for153C-WT Hv1: ϕ153C,153C = ϕWT,WT = 95, ϕ153C,WT = 23 ± 2. See text for details. (b) Model of opening of the two pores of Hv1. Cooperativity is quantified by the parameter ϕ, which is the ratio between the open probability of a given subunit when the neighboring subunit is in the open conformation and the open probability of the same subunit when the neighboring subunit is in the closed conformation. ϕ > 1 means that the opening of one pore makes it easier for the other pore to open (positive cooperativity). The scheme represents the case of a heterodimer made of subunit A (gray) and subunit B (white) with different voltage dependencies of activation (KA and KB). Subunit A can be 153C or MS, and subunit B can be WT or ZS. The transition from square shape to round shape represents the conformational change associated with opening (see also Supplementary Fig.4).
	Figure 5. Hv1 voltage-sensor coupling detected by voltage-clamp fluorometry. (a) Voltage sensor movement detected on Hv1 195C labeled with TAMRA-MTS. Changes in fluorescence (F) and proton current (I) were produced by the indicated voltage step (V) from −80 mV to +160 mV. Traces in gray were recorded at pHo 7.4. Traces in black were recorded from the same oocyte in acetate buffer at pHo 6.3, which also acidifies the internal solution (see text). (b–c) Comparison between voltage sensor movement in labeled 195C/195C homodimers and 195C/218S heterodimers. Heterodimers were produced by co-expressing the Hv1 195C subunit with an excess of the 218S subunit. Fluorescence changes and proton currents were elicited by voltage steps from −200 to +200 mV in 80 mV increments. Holding potential was −80 mV, pHo was 6.3. (d) Normalized F-Vs from labeled oocytes expressing the Hv1 195C subunit (red circles) or co-expressing the 195C and 218S subunits (black squares). Fluorescence was measured at the end of the 500-ms long test-pulse (voltage protocol and recording conditions as in b–c. Each point is the average of 5 measurements ± s.e.m.
	Figure 6. Proposed basic gating mechanism for the Hv1 channel. Opening of one subunit favors the opening of the neighboring subunit. As a result, the two subunits tend to be in the same state, either both closed or both open. With a cooperativity factor ϕ = 60 the total fraction of channels with only one open pore can never be higher than 12%. The represented states are connected by at least two transitions: a voltage sensing transition (black arrows) responsible for the fast component of sensor movement detected by VCF, and an opening transition (green arrows). Based on the similarity between the VSD of Hv1 and the VSDs of other voltage-gated channels, the voltage-sensing process is represented as upward movement of charges (shown in white). The C-termini of the two subunits are shown as coiled-coil.

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