Structural and biophysical determinants of single Ca(V)3.1 and Ca(V)3.2 T-type calcium channel inhibition by N(2)O.
We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels Ca(V)3.1 and Ca(V)3.2 by nitrous oxide (N(2)O). 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(2)O significantly inhibited Ca(V)3.2, and - less pronounced - Ca(V)3.1. A Ca(V)3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing < or = 3 channels N(2)O reduced average peak current of Ca(V)3.2 by decreasing open probability and open time duration. Effects on Ca(V)3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single Ca(V)3.1 channels were significantly less active than Ca(V)3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within Ca(V)3.2 (H191), are responsible for the subtype-prevalent N(2)O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N(2)O on Ca(V)3.2. 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(V)3.1.
Bartels
Peter
P
Department of Pharmacology, University of Cologne, 50931 Koeln, Germany.
Behnke
Kerstin
K
Michels
Guido
G
Groner
Ferdi
F
Schneider
Toni
T
Henry
Margit
M
Barrett
Paula Q
PQ
Kang
Ho-Won
HW
Lee
Jung-Ha
JH
Wiesen
Martin H J
MH
Matthes
Jan
J
Herzig
Stefan
S
D020747:Calcium Channels, T-Type / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D004594:Electrophysiology / Q000379:methods; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D009609:Nitrous Oxide / Q000378:metabolism / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism
C121855:CACNA1G protein, human; C479952:CACNA1H protein, human; D020747:Calcium Channels, T-Type; D011993:Recombinant Fusion Proteins; D009609:Nitrous Oxide