Structural and biophysical determinants of single Ca V3.1 and Ca V3.2 T-type calcium channel inhibition by N 2O

Abstract

We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels Ca V3.1 and Ca V3.2 by nitrous oxide (N 2O). To identify functionally important channel structures, chimeras with reciprocal exchange of the N-terminal domains I and II and C-terminal domains III and IV were examined. In whole-cell recordings N 2O significantly inhibited Ca V3.2, and – less pronounced – Ca V3.1. A Ca V3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing ≤3 channels N 2O reduced average peak current of Ca V3.2 by decreasing open probability and open time duration. Effects on Ca V3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single Ca V3.1 channels were significantly less active than Ca V3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within Ca V3.2 (H191), are responsible for the subtype-prevalent N 2O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N 2O on Ca V3.2. Such a fingerprint of single channels can help identifying the molecular nature of native channels. This is exemplified by a characterization of single channels expressed in human hMTC cells as functional homologues of recombinant Ca V3.1.