The amino side of the C-terminus determines fast inactivation of the T-type calcium channel alpha1G.
We analysed the kinetic properties of the fast inactivating T-type calcium channel alpha1G in HEK 293 cells transfected with different alpha1G chimeras, containing the N-terminus, III-IV linker or various C-terminal regions of the slowly inactivating L-type alpha1C. A highly negatively charged region of 23 amino acids at the amino side of the intracellular carboxy terminus of alpha1G was found to be critical for fast inactivation. The N-terminus of alpha1G does not seem to be necessary for inactivation of the T-type calcium channel because replacement of the a1G N-terminus with the alpha1C N-terminus did not influence channel kinetics at all. Replacing the III-IV linker of alpha1G with that of a1C decreased the rate of inactivation at -20 mV from 15.8 +/- 1.8 to 8.5 +/- 1.1 ms, and shifted the potential for half-maximal inactivation from -69.6 +/- 0.8 to -54.0 +/- 1.7 mV. However, these parameters were not significantly different at other potentials. We suggest a putative 'ball-and-chain'-like mechanism for inactivation in which the negative charges function as an acceptor domain for a ball, hypothetically located at a different intracellular part of the channel. In addition, transferring the IQ motif and EF hand of alpha1C to alpha1G does not confer Ca2+-dependent inactivation on alpha1G, suggesting that other sequences besides the C-terminus are needed for Ca2+-dependent inactivation of alpha1C.
Staes
M
M
Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, Belgium.
Talavera
K
K
Klugbauer
N
N
Prenen
J
J
Lacinova
L
L
Droogmans
G
G
Hofmann
F
F
Nilius
B
B
D000465:Algorithms; D001464:Barium / Q000378:metabolism; D001711:Biotransformation; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020747:Calcium Channels, T-Type / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D004594:Electrophysiology; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002121:Calcium Channel Blockers; D020747:Calcium Channels, T-Type; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D001464:Barium; D002118:Calcium