Bi‐directional pH dependency of CALHM1 ion channel

Calcium homeostasis modulator (CALHM) membrane proteins are nonselective ion channels drawing attention regarding their roles in modulating neuronal activity and taste sensation. CALHM1-expressing cells show voltage-gated slowly activating ionic currents. The voltage-dependence of CALHM1 is negatively affected by extracellular Ca2+ ([Ca2+ ]e ), while facilitated by temperature. Here we investigated the effect of extracellular and intracellular pH (pHe and pHi ) on the electrophysiological properties of CALHM1 overexpressed in CHO cells. When normalized to the control at pHe 7.4, the amplitudes of CALHM1 current (ICALHM1 ) were suppressed to 20 % at pHe 6.2, while increased to 500 % at pHe 8.6. In the whole-cell configuration of patch clamp, changing pHi from 7.4 to 6.2 or 8.6 showed the acid-induced inhibition and alkali-induced activation of ICALHM1 . The voltage-dependence of CALHM1 was shifted to the negative membrane potential at the alkaline pHe and pHi , while positive membrane potential at the acidic pHe and pHi . The pHi effect was also confirmed in the inside-out configuration of patch clamp. The open probability (Po ) of multiple CALHM1 channel decreased at pH 6.2, while increased at pH 8.6 compared with pH 7.4. To get a clue to identify the pH sensing amino acid residues, we utilized homology model performed with the SWISS-MODEL workspace, based on the cryo-EM structure of human CALHM2. Then we conducted site-directed mutagenesis of the water-accessible charged amino acids of CALHM1. Among 23 candidates, mutations at E17, K229, E233, D257 and E259 in the intracellular space reduced the alkali-induced facilitation of ICALHM1 , while not altered the acid-induced inhibition. According to the molecular structure model of killifish CALHM1 with octameric structure, the five residues located at N or C termini gathered closely in the intracellular space. Considering the large pore diameter of CALHM1 (~14 Å), we cautiously propose that the sensitivity to alkaline pHe could be because the protons transfer between hydronium ions in the pore. The study firstly demonstrated the remarkable pH-sensitivity of CALHM1 and its pH-sensing residues, which might be responsible for the modulation of neuronal excitability during dynamic changes of the regional pH in brain or sensory organs.