Abstract
The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca(V) channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca(V)1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly(432) and Gly(436) in Ca(V)1.2) to form a triglycine motif unique to HVA Ca(V) channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca(2+) channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca(V)1.2, alpha-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca(V)1.2 wild type > G770A > G422A congruent with G436A >> G432A (from the fastest to the slowest). Mutations at position Gly(432) produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly(436) to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX(3)G residues blunted Ca(2+)-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca(V)2.3, mutation of the distal glycine Gly(352) impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX(3)G motif in the voltage-dependent activation and inactivation gating of HVA Ca(V) channels with the distal glycine residue being mostly involved in the inactivation gating.
Highlights
Molecular cloning has identified the primary structures for 10 distinct calcium channel CaV␣1 subunits (1, 4 –9) that are classified into three main subfamilies according to their gating properties (CaV1, CaV2, and CaV3)
The closed KcsA structure, where the inner helices adopt an “inverted teepee” conformation crossing over near the intracellular surface at the helix bundle, would represent the closed state (10). Based on these static crystal structures, it was proposed that Kϩ channels could open by bending the pore-lining ␣-helix at this nearly universal glycine residue located in the middle of the pore lining helix (23)
The bottom alignments show the primary sequences of the IS6 and IIS6 segments of the rabbit L-type CaV1.2 (GenBankTM accession number X15539) and the human CaV2.3 (GenBankTM accession number L27745) channels used in this study
Summary
Recombinant DNA Techniques— cDNAs coding for wild-type rabbit CaV1.2 (GenBankTM X15539), wild-type human CaV2.3 (GenBankTM L27745) (31), and rat CaV3 (GenBankTM M88751) (32) were kindly donated by Drs E. The rat brain CaV ␣2b␦ subunit provided by Dr T. P. Snutch is DECEMBER 22, 2006 VOLUME 281 NUMBER 51. W enohpoules-ocoelclyctuesrrienntthsewperreesemnceeasoufrCedaVi␣n21b0dmanMdBCaa2Vϩt3hsrouubguhnoitus.t.TAhcetbivaactkiognroduantda (E0.5 act, z, and Vrev) were estimated from the mean I-V relationships and fitted to Boltzmann Equation 1. Inactivation properties were estimated from the fraction of noninactivating currents at 800 ms or r800 values. R800 values could not be determined for mutants with “time to peak” values of Ͼ500 ms. Peak IBa was determined from the peak I-V relationships for the corresponding experiments. The data are shown with the mean Ϯ S.E. of the individual experiments, and the number of experiments appears in parentheses
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