Molecular determinants of inactivation within the I-II linker of alpha 1E (Ca(v)2.3) calcium channels

Voltage-dependent inactivation of Ca(v)2.3 channels was investigated using point mutations in the beta -subunit-binding site (AID) of the I-II linker. The quintuple mutant alpha 1E N381K + R384L + A385D + D388T + K389Q (NRADK-KLDTQ) inactivated like the wild-type alpha 1E. In contrast, mutations of alpha 1E at position R378 (position 5 of AID) into negatively charged residues Glu (E) or Asp (D) significantly slowed inactivation kinetics and shifted the voltage dependence of inactivation to more positive voltages. When co-injected with beta3, R378E inactivated with T-Inact = 538 +/- 54 ms (n = 14) as compared with 74 +/- 4 ms (n = 21) for alpha 1E (p < 0.001) with a mid-potential of inactivation E-0.5 = -44 +/- 2 mV (n = 10) for R378E as compared with E-0.5 = -64 +/- 3 mV (n = 9) for 1E. A series of mutations at position R378 suggest that positively charged residues could promote voltage-dependent inactivation. R378K behaved like the wild-type alpha 1E whereas R378Q displayed intermediate inactivation kinetics. The reverse mutation E462R in the L-type alpha 1C (Ca(v)1.2) produced channels with inactivation properties comparable to alpha 1E R378E, Hence, position 5 of the AID motif in the I-II linker could play a significant role in the inactivation of Ca(v)1.2 and Ca(v)2.3 channels.