Double mutant cycle analysis identified a critical leucine residue in the IIS4S5 linker for the activation of the Ca v2.3 calcium channel

Abstract

By analogy with K V channels, we tested the hypothesis that channel activation involves electromechanical coupling between S6 and the S4S5 linker in Ca V2.3. Among the 11 positions tested in the S4S5 linker of domain II, mutations of the leucine residue at position 596 were found to destabilize significantly the closed state with a -50 mV shift in the activation potential and a -20mV shift in its charge-voltage relationship as compared with Ca V2.3 wt. A double mutant cycle analysis was performed by introducing pairs of glycine residues between S4S5 and S6 of Domain II. Strong coupling energies (ΔΔG interact > 2 kcal mol -1) were measured for the activation gating of 12 of 39 pairs of mutants. Leu-596 (IIS4S5) was strongly coupled with distal residues in IIS6 from Leu-699 to Asp-704. In particular, the double mutant L596G/I701G showed strong cooperativity with a ΔΔG interact ≈6 kcal mol -1suggesting that both positions contribute to the activation gating of the channel. Altogether, our results highlight the role of a leucine residue in S4S5 and provide the first series of evidence that the IIS4S5 and IIS6 regions are energetically coupled during the activation of a voltage-gated Ca v channel. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.