Function modulation of MDSC mHv1 channel by N terminal length.

Abstract

Voltage-gated proton channel (Hv1) regulate the immunosuppressive function of myeloid-derived suppressor cells (MDSC) in mice. We asked if there were Hv1 isoforms present in MDSC. A prediction made by the NCBI showed that there could be longer isoforms of mouse Hv1, of 42 and four extra amino acid residues, called mHv1.2 and mHv1.3, respectively. These additional residues are predicted to express at the very beginning of the N terminal domain. We validated the prediction in vitro by RT-PCR, founding two isoforms of mHv1 and cloned them. We expressed these isoforms into Xenopus laevis oocytes for proton current recording studies by macropatch voltage clamp. All these isoforms studied satisfy the typical proton channel properties regarding the selectivity and channel activation kinetics. However, these isoforms exhibited clear differences both in their voltage-dependent gating and kinetics: the longer isoform, mHv1.2 exhibited the most right-displaced V0.5, followed by mHv1.3 and mHv1.1, while the most accelerated opening kinetics belonged to mHv1.1, followed by mHv1.3 and finally mHv1.2. We studied the unitary conductance of these isoforms by non-stationary noise analysis, evidencing values related to the fS order. All the cloned isoforms are expressed in the membrane as dimers, as shown by the estimation of the charge coupled to the opening by limit slope technique. These results strongly suggest that all these isoforms were typical Hv1 channels that possess unique biophysical properties. Since the different isoforms exhibited exclusively differences in their N terminal, the longer the N terminal length, the slower the opening kinetics and the shifted to the right the GV curve. We propose that the differences in the voltage-dependent gating exhibited by the isoforms are due by the extension of their N terminal.