Supplementary Figure 1: State-dependent modification of S1 residues.
From: A specialized molecular motion opens the Hv1 voltage-gated proton channel
(a) WT channels undergo current rundown after successive depolarizations to +60 mV in TEVC. (i) Cartoon of Hv1. (ii) WT TEVC currents elicited by a +60 mV voltage step at the beginning (grey) and at the end (black) of protocols used to measure external MTS effects with 10% and 60% of the time at +60 mV. (iii) Average current rundown of WT channels spending either 10% or 60% of the time at +60 mV. Rundown is expressed as the ratio of current (arrowhead in (ii)) at the end of the protocol on current at the beginning of the protocol (n = 4 oocytes for each protocol).
(b-c) Extracellular MTSES (b) or MTSET (c) have no effect on WT channels. (i) Cartoon of Hv1 with bath application of MTSES or MTSET (red). (ii) WT TEVC currents elicited by a +60 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSES or MTSET. (iii) Steady-state MTS-induced inhibition measured at the arrowhead in (ii) for WT channels spending either 10% or 60% of the time at +60 mV (MTSES, n = 4 oocytes for each protocol; MTSET, n = 5 oocytes for each protocol). Note that the amount of current decrease after MTS treatment and washout corresponds to the amount of natural current rundown during the same protocol (Supplementary Fig. 1a).
(d) D171C is more rapidly modified by external MTSET at positive voltage. (i) Cartoon shows D171C modified by extracellular MTSET. (ii) D171C currents in response to a voltage step to +60 mV before (grey trace) and after (black trace) application of 1 mM MTSET and washout. (iii) Steady-state change in current amplitude measured at arrowhead in (ii) after MTSET application and washout with 10 or 60% of the time at +60 mV. (iv) Rate constants of D171C modification with 10% (kMTSET = 37 ± 6 M−1 s−1, n = 9 oocytes) or 60% (kMTSET = 83 ± 13 M−1 s−1, n = 9 oocytes) of the time at +60 mV. p < 0.01, two-tailed Student’s t-test.
(e) Intracellular MTSET has no effect on WT channels (n = 4 patches). (i) Cartoon of Hv1 with bath application of MTSET. (ii) WT currents from inside-out patches elicited by a +80 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSET. (iii) Steady-state inhibition of wt channels after application and washout of 1 mM MTSET, measured at arrowhead in (ii).
(f) Intracellular MTSET slows down the rate of deactivation of I150C. (i) Cartoon shows I150C modified by intracellular MTSET. (ii) I150C currents elicited by a +80 mV voltage step before (grey) and after (black) application and washout of 1 mM MTSET.
(g) Intracellular MTSET accelerates opening of I154C at positive voltage (n = 4 patches). (i) Cartoon shows I154C modified by intracellular MTSET. (ii) Currents before and after application of 1 mM MTSET, either at −80 mV, or during repeated steps to +80 mV (chronological color code from violet to red), followed by washout at −80 mV. Inset, magnification of the current traces at stimulus onset. (iii) Steady-state increase in current amplitude at arrowhead in (ii) after application and washout of MTSET at −80 or +80 mV (p < 0.05, two-tailed Student’s t-test). Dashed line represents absence of effect. (iv) Time course of MTSET effect at arrowhead in (ii), at −80 (linear fit) or +80 mV (single exponential fit). (v) Rate constants of I154C modification at −80 mV (kMTSET = 0.79 ± 0.16 M−1 s−1) or +80 mV (kMTSET = 56 ± 18 M−1 s−1) measured from the fits in (iv).
(h) V157C is modified by internal MTSET at positive voltage (n = 3 patches). (i) Cartoon shows V157C modified by intracellular MTSET. (ii) Currents before and after application of 0.5 mM MTSET, either at −80 mV, or during repeated steps to +80 mV (chronological color code from violet to red), followed by washout at −80 mV. Inset, magnification of the current traces at stimulus onset. (iii) Steady-state increase in current amplitude at arrowhead in (ii) after application and washout of MTSET at −80 or +80 mV. Dashed line represents absence of effect. (iv) Time course of MTSET effect at arrowhead in (ii), at −80 or +80 mV. (v) Rate constants of V157C modification at −80 mV (kMTSET = 0.74 ± 0.06 M−1 s−1) or +80 mV (kMTSET = 150 ± 72 M−1 s−1).
Error bars, s.e.m.
