CHIMERIC L-TYPE CA2+ CHANNELS EXPRESSED IN XENOPUS-LAEVIS OOCYTES REVEAL ROLE OF REPEAT-III AND REPEAT-IV IN ACTIVATION GATING

1. Chimeric alpha(1) subunits consisting of repeat I and II from the rabbit cardiac (alpha(1C-a)) and repeat III and IV from the carp skeletal muscle Ca2+ channel (alpha(1s)) were constructed and expressed in Xenopus laevis oocytes without co-expressing other channel subunits. Ba2+-current kinetics of five chimeric channel constructs were studied in Xenopus oocytes using the two-microelectrode technique. 2. Exchange of repeats III and IV of alpha(1C-a) with sequences of alpha(1S) results in a significantly slower and biexponential activation (apparent activation time constants tau(1act) = 19.8 +/- 1.8 ms and tau(2act) = 214 +/- 28.7 ms, n = 7) of expressed Ca2+ channel currents; no current inactivation was observable during an 800 ms test pulse to 0 mV. 3. Activation of a chimera consisting of repeats I, II and IV from the alpha(1C-a) subunit and repeat III from alpha(1S) was fast and monoexponential (tau(iact) = 6.33 +/- 1.7 ms, n = 5) and the current inactivated during a 350 ms test pulse to 0 mV (tau(inact) = 175 +/- 22 ms, n = 5). The current kinetics of this construct did not significantly differ from kinetics of a construct consisting of repeats I to IV from alpha(1C-a) (tau(1act) = 6.6 + 2.1 ms; tau(inact) = 198 +/- 14 ms; n = 9). 4. Expression of a chimera where only the region IIIS6 to IVS5 and adjacent amino acids were changed from the cardiac alpha(1C-a) to alpha(1S) yielded channels that activated slowly at 0 mV with a biexponential time course (apparent tau(1act) = 12.9 +/- 1.9 ms and tau(2act) = 262 +/- 18.9 ms, n = 6). 5. Single channel conductances of a fast (19.4 +/- 1.1 pS) and a slow activating chimera (18.6 +/- 1.2 pS) were estimated with 90 mM Ba2+ as charge carrier to be close to the conductance of L-type Ca2+ channels. 6. These results demonstrate that repeats III and IV of alpha(1S) from carp skeletal muscle possess structural determinants for 'slow activation' of L-type Ca2+ channels.