Identification of an HV1 voltage-gated proton channel in insects

Abstract

The voltage-gated proton channel 1 (HV1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV1 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 HV1 channel. In the present study, we report the existence of an HV1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV1 orthologues in various polyneopteran insects. To confirm that these putative HV1 orthologues were functional channels, we studied the HV1 channel of Nicoletia phytophila (NpHV1), an insect of the Zygentoma order, in more detail. NpHV1 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 pH- and voltage-dependent gating. Interestingly, NpHV1 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. Database Nucleotide sequence data have been deposited in the GenBank database under accession number KT780722. The HV1 voltage-gated proton channel extrudes cellular protons and compensates charge during the respiratory burst. In contrast to a postulated dogma, we report the existence of HV1 in insects. NpHV1 from Nicoletia phytophila is proton selectivity, and shows pH- and voltage-dependent gating. Substitutions of an aspartate in S1 leads to a loss of proton-selective conduction, confirming its importance as selectivity filter in all HV1s.