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

MANUEL LLANOS; CLARA VENTURA; PEDRO MARTÍN; NICOLAS ENRIQUE; JUAN IGNACIO FELICE; LUCIANA GAVERNET; VERONICA MILESI

JOURNAL OF CHEMICAL INFORMATION AND MODELING

AMER CHEMICAL SOC

Lugar: Washington; Año: 2022

1549-9596

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 multi-template 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. Further GaMD simulations of the ATP-bound dimeric hHv1 model showed that ATP effectively interacts with a cluster of positively charged residues from the cytoplasmic N and C terminal S1 and S4 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 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 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.