Fig. 4: Alb binds to S3-S4 loop of hHv1 channels.

hHv1, CiHv1, hHv1-G199L and chimeric channels were expressed in HEK293T cells and studied by whole-cell patch clamp. a Top, sequence alignments of the S1-S2 and S3-S4 motifs of hHv1 (black), CiHv1 (blue) and loop-transplant chimeras. Bottom, proton current traces at 0 mV for channels in the absence (black trace) or presence of 75 µM Alb (red trace). b Activation by Alb (75 μM) of WT and chimeric channels, current at the end of a test pulse to 0 mV is plotted. Values are normalized to current amplitude before Alb application. The cartoons represent the topology of a single subunit with hHv1 segments in black and CiHv1 segments in blue, n = 3–6 cells. c Mutagenesis scan of the S3-S4 loop of hHv1 (Cys substitution except for V187A, L189A, and L203A) showing changes with 75 µM Alb at 0 mV; numbering corresponds to the amino acid sequence of hHv1. Values normalized to the current amplitude before Alb application, n = 3–6 cells. d Left, proton current traces for hHv1-G199L channels in the absence (up) and presence of 1 µM Alb (bottom) with steps of 20 mV from −60 mV to +40 mV. Right, Alb dose-response relationship for hHv1-G199L provides an EC₅₀ for Alb activation of 3.4 ± 0.5 µM estimated by a fit to the Hill relationship with a coefficient of 0.93 ± 0.10. Values are normalized to mean proton current amplitude in the absence of Alb. Some error bars are smaller than symbols, n = 3–8 cells. e Cartoon of one TAMRA-Alb molecule binding to the two subunits in a hHv1-G199L-TFP channel. f Left, incubation of TAMRA-Alb (red) with hHv1-G199L-TFP channels (teal) produces single colocalized particles (white) with both TAMRA and TFP fluorescence at the surface of cells (yellow circles). Middle, montage of photobleaching time course of a single fluorescent particle (arrow in left panel) during continuous excitation to bleach the fluorophores (every fifth frame is shown). Right, time courses for simultaneous photobleaching of both fluorophores in the particle, showing one stepwise change in fluorescence intensity for TAMRA-Alb and two for hHv1-G199L-TFP (arrows). g Histogram of photobleaching steps for hHv1-G199L-TFP (teal bars) and TAMRA-Alb (red bars) on simultaneous photobleaching. 89% of studied particles with hHv1-TFP were bleached in 2 steps. Data analyzed by the approach of Hines⁶⁷ estimates two hHv1-G199L subunits in surface particles with a confidence of >0.999 (Supplementary Table 2). Among all particles containing both fluorescent colors 95% had one TAMRA-Alb bleaching step at 3 µM TAMRA-Alb (Supplementary Table 3). Values are mean ± SEM. Source data are provided in the Source Data file.