Identification Of Activating And Inhibiting Gating Modifier Toxins That Target The S3-S4 Loop Of The Human Proton Channel, Hhv1

Human Hv1 channels (hHv1) are implicated in a wide variety of cellular functions including reactive oxygen species production, sperm capacitation and cell proliferation in cancer. Each channel has two hHv1 subunits that are homologous to the S1-S4 voltage sensing domains (VSD) in voltage-gated K+ and Na+ channels. The S3-S4 loop of VSD is a hot spot for interaction of known gating modifier toxins, which either activate or inhibit by stabilizing the VSD in different conformations. In the accompanying abstract (Kennedy, Zhao, et al.), we describe isolation of C5 and C6 from ∼1 million member phage display library constructed on an inhibitor cysteine knot (ICK) scaffold, a backbone found in many gating modifier neurotoxins. As expected, the S3-S4 loop of hHv1 was found to be the major binding epitope for C6 toxin. Whereas, ci-Hv1 channels from sea squirt were insensitive to 1 μM C6, transplanting the hHv1 S3b-S4a motif to ci-Hv1 conferred the chimera with sensitivity (maximal blockade 70%; half maximal inhibition at 35 nM); the hHv1 motif was also able to confer C6 blockade to otherwise insensitive Kv2.1. Scanning the motif in hHv1 by Cys scanning across 12 residues revealed four sites that decreased affinity of C6 over 3-fold, one site that increased inhibition 75-fold (and permits 100% inhibition), and one site that allows C6 binding to activate the channel by 30% (half maximal activation at 250 nM). Consistent with a shared VSD trapping mechanism, C6 inhibits whereas C5 activates, yet both modify the kinetics of activation and deactivation as well as the voltage-dependence of gating, and while they are 39 and 41 residues, respectively, the C-terminal 31 residues are identical. A tethered-toxin strategy has been employed to map the C6 interaction surface with hHv1 as a prelude to designing more potent inhibitors and activators; ten important residues that are charged or polar have been identified.