(948.6) Bi-directional pH dependency of CALHM1 ion channel

Poster Board Number: E508

Jae Won Kwon (Seoul National University, Seoul National University), Young Keul Jeon (Seoul National University), Sung Joon Kim (Seoul National University)

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.

Support or Funding Information

This work was supported by grants from the National Research Foundation of Korea (NRF- 2021R1A2C2007243).