Identification of an H_V1 voltage‐gated proton channel in insects

Abstract

The voltage-gated proton channel 1 (H V 1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. H V 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an H V 1 channel. In the present study, we report the existence of an H V 1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative H V 1 orthologues in various polyneopteran insects. To confirm that these putative H V 1 orthologues were functional channels, we studied the H V 1 channel of Nicoletia phytophila (NpH V 1), an insect of the Zygentoma order, in more detail. NpH V 1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pHand voltage-dependent gating. Interestingly, NpH V 1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels.

Comparison between mouse and sea urchin orthologs of voltage-gated proton channel suggests role of S3 segment in activation gating

“…2A), indicating that SpHv1 is highly selective to proton as established in previously characterized Hv1 channels [19][20][21][22][23].…”

“…Diversity of activation kinetics between Hv1 orthologs 21 A-D. representative current traces of SpHv1-background S3a chimera series with replacement of only S3a(A), S3a and N terminus plus S1(B), S3a and S2 (C), S3a and S4(D) by the corresponding segment from mHv1. Holding potential was -60 mV.…”

“…A gene encoding a voltage-gated proton channel, HVCN1, has been identified from human, mouse, ascidian [19,20], insect [21], dinoflagellates [22], marine phytoplankton [23] and in other metazoans. As molecular architecture, Hv1 lacks an authentic pore domain and the voltage sensor domain is responsible for both voltage sensing and proton permeation.…”

The intimate and controversial relationship between voltage‐gated proton channels and the phagocyte NADPH oxidase

“…Finally, knowing the gene enables the identification of H V 1 in diverse species. H V 1 genes that have been confirmed by voltage‐clamp studies in heterologous expression systems include: human , mouse, and sea squirt Ciona intestinalis , dinoflagellates Karlodinium veneficum and Lingulodinium polyedrum , coccolithophores Emiliania huxleyi and Coccolithus pelagicus , diatom Phaeodactylum tricornutum , insect Nicoletia phytophila , and sea urchin Strongylocentrotus purpuratus .…”

Hv1 Proton Channels in Dinoflagellates: Not Just for Bioluminescence?

“…The exquisite proton selectivity of H V 1 arises from a critical Asp in S1 (Chaves et al. ; Musset et al. ; Smith et al.…”