Channelrhodopsin is a light-gated cation channel whose reaction cycle involves proton-transfer reactions. Understanding how channelrhodopsin works is 16a Sunday,

H v 1 voltage-gated proton channels appear to conduct H þ through a voltage sensor domain (VSD) that is homologous to that found in voltage-dependent cation channels and phosphatases. A conserved S4 transmembrane helix that contains a series of at least three Arg residues is integral to the voltage sensing function of all VSD proteins. In contrast to other VSD-containing proteins, voltagegated proton channels possess an additional unique biophysical property: coupling of the transmembrane pH gradient to voltage dependent activation. For both native voltage-gated H þ currents and expressed H v 1 channels, the apparent voltage threshold for H þ current activation (V thr ) shifts linearly ~40 mV per log([H þ ]) over at least five pH units. The molecular mechanism of coupling between voltage and the pH gradients represents one of the central mysteries of proton channel function. What constitutes the pH sensor in proton channels and how does it interact with the voltage sensor? DeCoursey and colleagues previously proposed a model for H þ channel gating wherein protonation of discrete sites that are alternatively exposed to either the extra-or intra-cellular milieu regulates the voltage-dependence of channel opening (Cherny et al., 1996); the required first step in this model is deprotonation of an extracellular H þ binding site. In order to identify residues that mediate pH-dependent regulation of voltage sensitivity in H v 1, we performed sitedirected mutagenesis to convert each of the candidate H þ acceptors in the H v 1 VSD to either neutral (alanine or asparagine), basic (arginine) or H þ -titratable (histidine) amino acids. Mutant channels were expressed in HEK-293 cells and V thr was determined under a variety of imposed pH gradients using whole-cell voltage clamp. Surprisingly, charge-neutralizing mutations failed to abrogate pH gradient sensing in H v 1. Our findings are interpreted in the context of the Cherny and DeCoursey model for proton channel gating.

In contrast, at such negative voltages, Hþ at pH ¼ 6.2 was much less effective in increasing open probability, leading to the estimated C value ¼ ~1.3, placing Hþ between Ca2þ and Mg2þ in the ability to promote opening in the absence of voltage sensor activation. Likewise, at the respective saturation concentrations, Hþ was less effective than Ca2þ at regulating channel kinetics. Our electrophysiological measurements and simulations collectively suggest that Hþ is a partial agonist of the RCK1 high-affinity Ca2þ sensor and that a small change in the relative position of His and Asp residues in the sensor, ~0.1 nm, may underlie the activation of the channel by Hþ. Supported by NIH.

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TvoK is a prokaryotic K þ channel whose gating is modulated by divalent cation-binding to a carboxy-terminal RCK domain. To gain insight toward mechanisms underlying divalent cation binding and subsequent conformational changes, we measured chemical shift perturbations upon ligand binding in the soluble cytoplasmic RCK domain of TvoK using heteronuclear NMR spectroscopy.Uniformly 15 N-labeled, highly deuterated TvoK RCK domain was overexpressed in E.coli and purified by affinity and gel-filtration chromatography. 15 N-HSQC spectra showed well-dispersed crosspeaks corresponding to >85% of the 238 predicted backbone NH groups. Five-point titration experiments using 0 to 100 ˛1/4M Mn 2þ identified 12 residues that surrounded a putative divalent cation binding site, as indicated by spectral line-broadening due to the paramagnetic relaxation enhancement effect of Mn 2þ (Mn-PRE). Partial resonance assignments, made through a combination of HNCA experiments and residue-specific 15 N-labeling, identify D192 and E226 as key residues in divalent cation coordination, as indicated by high sensitivity to Mn-PRE (K app < 10 ˛1/4M). Further resonance assignments will identify remaining residues that lie within ~15 A ˚of the binding site. These experiments may reveal differences between the structural and chemical properties of the TvoK binding site and the Ca 2þ -selective binding site of the MthK RCK domain, which may underlie differential selectivities of MthK and TvoK RCK domains for divalent cations.