Novel Dimeric hHv1 Model and Structural Bioinformatic Analysis Reveal an ATP-Binding Site Resulting in a Channel Activating Effect

Abstract

The human voltage-gated proton channel (hHv1) is a highly selective ion channel codified by the HVCN1 gene. It plays a fundamental role in several physiological processes such as innate and adaptive immunity, insulin secretion, and sperm capacitation. Moreover, in humans, a higher hHv1 expression/function has been reported in several types of cancer cells. Here we report a multitemplate homology model of the hHv1 channel, built and refined as a dimer in Rosetta. The model was then subjected to extensive Gaussian accelerated molecular dynamics (GaMD) for enhanced conformational sampling, and representative snapshots were extracted by clustering analysis. Combining different structure-and sequence-based methodologies, we predicted a putative ATP-binding site located on the intracellular portion of the channel. Furthermore, GaMD simulations of the ATP-bound dimeric hHv1 model showed that ATP interacts with a cluster of positively charged residues from the cytoplasmic N and C terminal segments. According to the in silico predictions, we found that 3 mM intracellular ATP significantly increases the H + current mediated by the hHv1 channel expressed in HEK293 cells and measured by the patch-clamp technique in an inside-out configuration (2.86 ± 0.63 fold over control at +40 mV). When ATP was added on the extracellular side, it was not able to activate the channel supporting the idea that the ATP-binding site resides in the intracellular face of the hHV1 channel. In a physiological and pathophysiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H + efflux, especially in cells where hHv1 channels are relevant for pH regulation, such as pancreatic β-cells, immune cells, and cancer cells.

ATP modulates the activity of the voltage‐gated proton channel through direct binding interaction

“…R219A) by using one‐way ANOVA with post hoc Tukey–Kramer test]. This result was inconsistent with a previously reported result (Llanos et al., 2022), which showed a slight effect of ATP on the R223A mutant of human‐Hv1. These inconsistencies may be attributed to the following: (1) we used mHv1 in this study, while they used human‐Hv1; and (2) multiple ATP‐binding sites may exist, and single‐point mutation experiments do not account for this possibility.…”

“…Although no significant difference was observed, a slightly weaker effect was noted in the R219A mutant than in the wild type (WT) [P = 0.0641 (WT vs. R219A) by using one-way ANOVA with post hoc Tukey-Kramer test]. This result was inconsistent with a previously reported result (Llanos et al, 2022), which showed a slight effect of ATP on the R223A mutant of human-Hv1. These inconsistencies may be attributed to the following: (1) we used mHv1 in this study, while they used human-Hv1; and…”

“…A recent study conducted using a combination of molecular dynamics simulation and mutagenesis (Llanos et al., 2022) showed that ATP binds to the Hv1 dimer interface at the proximal end of the coiled‐coil region for S4. Through electrophysiological analysis, the study further confirmed that the ATP‐induced increase in the current amplitude was not observed in the human‐Hv1 mutant carrying an alanine substitution on Arg223.…”

“…One report suggested that ATP maintains proton channel activity in microglia (Morihata et al., 2000). Another showed that ATP influences the Hv1 current and suggested the presence of an ATP‐binding site in Hv1 through a computational approach (Llanos et al., 2022). However, the authors did not elaborate on the molecular mechanisms underlying these effects of ATP on Hv1 activity.…”

CHARMM-GUI Membrane Builder: Past, Current, and Future Developments and Applications

“…Another important aspect about the ion channel is drug binding. MD simulations are used to explore ligand binding stability and to better assess the induced-fit effect from docking results , or to verify the binding poses from structural biology works. , Ligand-binding free energy calculations yield a quantitative profile for comparison with experimental measurements . In addition, the dynamical effects exerted by ligands can be accessed from simulation trajectories.…”