Structural and biophysical determinants of single CaV3.1 and CaV3.2 T-type calcium channel inhibition by N2O

We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels CaV3.1 and CaV3.2 by nitrous oxide (N2O). 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 N2O significantly inhibited CaV3.2, and – less pronounced – CaV3.1. A CaV3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing ≤3 channels N2O reduced average peak current of CaV3.2 by decreasing open probability and open time duration. Effects on CaV3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single CaV3.1 channels were significantly less active than CaV3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within CaV3.2 (H191), are responsible for the subtype-prevalent N2O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N2O on CaV3.2. 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 CaV3.1.