The transient receptor potential vanilloid 1 ion channel is responsible for the perception of high temperatures and low extracellular pH, and it is also involved in the response to some pungent compounds. Importantly, it is also associated with the perception of pain and noxious stimuli. Here, we attempt to discern the molecular organization and location of the N and C termini of the transient receptor potential vanilloid 1 ion channel by measuring FRET between genetically attached enhanced yellow and cyan fluorescent protein to the N or C terminus of the channel protein, expressed in transfected HEK 293 cells or Xenopus laevis oocytes. The static measurements of the domain organization were mapped into an available cryo-electron microscopy density of the channel with good agreement. These measurements also provide novel insights into the organization of terminal domains and their proximity to the plasma membrane.

Phosphatidylinositol 4,5-bisphosphate (PIP₂) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), K_v7) K⁺ channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP₂ and performed extensive mutagenesis in regions suggested to be involved in PIP₂ interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP₂ as a probe, we found that PIP₂ regulates KCNQ3 channels through four different domains: 1) the A–B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2–S3 linker, and 4) the S4–S5 linker. We also found that the apparent strength of PIP₂ interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP₂ were consistent with the experimental data. Our results indicate that PIP₂ modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.

Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this work, we directly measure the proton depletion caused by activation of Hv1 proton channels using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Our results provide direct evidence of local proton depletion caused by flux through the proton-selective channel.

The voltage sensor (VS) domain in Hv1 proton channels mediates a voltage-dependent and H⁺-selective “aqueous” conductance (G_AQ) that is potently modulated by extracellular Zn²⁺. Although two conserved His residues are required for Zn²⁺ effects on G_AQ gating, the atomic structure of the Zn²⁺ coordination site and mechanism by which extracellular Zn²⁺ stabilizes a closed-state conformation remain unknown. Here we use His mutagenesis to identify residues that increase Zn²⁺ potency and are therefore likely to participate in first solvation shell interactions with Zn²⁺. Experimental Zn²⁺-mapping data were then used to constrain the structure of a new resting-state Hv1 model (Hv1 F). Molecular dynamics (MD) simulations show how protein and water atoms directly contribute to octahedral Zn²⁺ coordination spheres in Zn²⁺-bound and -unbound Hv1 F models. During MD simulations, we observed correlated movements of Zn²⁺-interacting side chains and residues in a highly conserved intracellular Coulombic network (ICN) that contains highly conserved Arg “gating charges” in S4 as well as acidic “counter-charges” in S2 and S3 and is known to control VS activation, suggesting that occupancy of the extracellular Zn²⁺ site is conformationally coupled to reorganization of the ICN. To test this hypothesis, we neutralized an ICN Glu residue (E153) and show that in addition to shifting G_AQ activation to more negative voltages, E153A also decreases Zn²⁺ potency. We speculate that extracellular gating-modifier toxins and other ligands may use a generally similar long-range conformational coupling mechanism to modulate VS activation in related ion channel proteins.