Molecular determinants of inhibition of the human proton channel hHv1 by the designer peptide C6 and a bivalent derivative

Abstract

Significance We designed C6 peptide to address the absence of specific inhibitors of human voltage-gated proton channels (hHv1). Two C6 bind to the two hHv1 voltage sensors at the resting state, inhibiting activation on depolarization. Here, we identify the C6–hHv1 binding interface using tethered-toxin variants and channel mutants, unveil an important role for negatively charged lipids, and present a model of the C6–hHv1 complex. Inspired by nature, we create a peptide with two C6 epitopes (C6 2 ) that binds to both channel subunits simultaneously, yielding picomolar affinity and significantly improved inhibition at high potentials. C6 and C6 2 are peptides designed to regulate hHv1, a channel involved in innate immune-system inflammatory pathophysiology, sperm capacitation, cancer-cell proliferation, and tissue damage in ischemic stroke.

Protection from acute lung injury by a peptide designed to inhibit the voltage-gated proton channel

“… 17 Since we isolated C6 to inhibit human Hv1, 8 it was important to determine that C6 also effectively blocked mouse Hv1 (mHv1) to validate conclusions drawn from the in vivo studies. Consistent with the same voltage sensor-trapping mechanism described for the human channel, 8 , 11 C6 decreased outward H + currents through mHv1 channels expressed in HEK293T cells in response to depolarizing steps measured by whole-cell patch clamp recording ( Figure 6 A) by shifting the half-maximal activation voltage (V 1/2 ) of the channel to more positive potentials ( Figure 6 B). C6 inhibition of mHv1 was dose-dependent, showing an inhibitory constant K i of 4.1 ± 0.3 nM and a Hill coefficient of 1.3 ± 0.1 at 0 mV as determined by fitting the dose-response curve to a Hill relationship ( Figure 6 C and STAR Methods ).…”

“…Thus, C6 was observed to be 3-fold less potent on mHv1 than on hHv1 ( K i = 1.5 nM at 0 mV). 11 This similar potency was expected given that the residues that bind C6 are homologous in the two channels ( Figures S1 A and S1C). Rationalizing why both low and high doses of C6 administered to the mice had significant effects on the LPS-treated mice ( Figures 1 and 2 ), C6 at 20 nM decreased mHv1 currents by ∼82%, shifting V 1/2 of the channel ∼24 mV ( Figures 6 A and 6B), and 2 μM C6 decreased the currents ∼95% ( Figure 6 C), shifting V 1/2 by ∼35 mV.…”

“…C6 binding is more avid at hyperpolarized voltages, like the resting membrane potential of neutrophils (∼−60 mV), when the Hv1 sensors are in the “down” conformation; binding therefore favors the channel closed state yielding an effective K i ∼ 1.5 nM at 0 mV. 8 , 11 Here, we study mouse Hv1 observing the C6 block to be like that for the human variant, but 3-fold less effective ( K i ∼ 4.1 nM at 0 mV), presumably a reflection of the primary sequences of their S3-S4 loops where C6 binds that differ at only three residues and are 78% identical ( Figure S1 C). Supporting a high degree of discrimination, 1 μM C6 did not inhibit the mammalian K + , Na + , and Ca 2+ channels we tested nor Hv1 from the sea squirt Ciona intestinalis despite its homology to mHv1 and hHv1.…”

“… 43 An advance specific to C6 that we recently achieved is an engineered form that is significantly more potent in vitro because it is bivalent (C6 2 ) and binds to both of its sites on Hv1 simultaneously. 11 We plan to study the pharmacokinetics of C6 and C6 2 to inform the consideration of the size and timing of doses and if modifications are required for therapeutic efficacy.…”

“… 8 C6 inhibits hHv1 with an equilibrium affinity of 1.5 nM at 0 mV, binding with positive cooperativity to the two voltage sensors in the channel and holding them in the down conformation that favors channel closure ( Figure S1 A). 8 , 11 …”