The Role of the GX9GX3G Motif in the Gating of High Voltage-activated Ca2+ Channels

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, ␣-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 Х G436A Ͼ Ͼ 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.Voltage-dependent calcium channels are membrane-bound proteins that form large aqueous pores for the selective diffusion of Ca 2ϩ ions across the plasma membrane (1, 2). Native Ca 2ϩ channels are composed of the pore-forming Ca V ␣1, the disulfur-linked dimer Ca V ␣2␦, the intracellular Ca V ␤ subunits (␤1-␤4), and in some cases the Ca V ␥ subunit (3). To date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca V ␣ 1 subunits (1, 4 -9) that are classified into three main subfamilies according to their gating properties (Ca V 1, Ca V 2, and Ca V 3). Whereas all voltage-gated Ca 2ϩ channel ␣1 subunits activate and inactivate in response to membrane depolarization, the high voltage-activated (HVA) 2 Ca V 1 and Ca V 2 ␣1 subunits operate at markedly more positive membrane potentials than low voltage-activated Ca V 3 channel ␣1 subunits.In the absence of a crystal structure for these proteins, details regarding the structural determinants of the channel inner pore as well as the molecular mechanism underlying the activation of Ca V ␣1 subunits remain sketchy. Structural studies have revealed that the architecture of the ion-selective pore is conserved in the homologous ␣ subunit of different K ϩ channels (10 -15) with the ...

CaV1.2 I-II linker structure and Timothy syndrome

“…Interestingly, homologous mutations decelerate inactivation kinetics in both Ca V 2.1 6 and Ca V 2.3. 3 Likewise, in our study, Ca V 2.2 G357R displayed extremely slow kinetics and a large hyperpolarizing shift in the voltage dependence of activation. In the case of Cav2.2, the mutation clearly G449R, appeared smaller than that of both single mutants (Fig.…”

“…A well-studied mutation of this glycine to almost all other amino acids is known to decelerate VDI drastically. [2][3][4][5][6][7] In humans, this mutation (G406R in human Ca V 1.2) results in a rare multisystem disorder known as Timothy syndrome, characterized by physical abnormalities, along with developmental and neurological deficiencies. 2,5 To explore whether this mutation may be mechanistically related to G449R, where we observed greater PL helicity and slowing of both VDI and CDI, we tested G436R alone and in combination with G449R electrophysiologically.…”

Double Mutant Cycle Analysis Identified a Critical Leucine Residue in the IIS4S5 Linker for the Activation of the CaV2.3 Calcium Channel

“…Whole Cell Recording and Data Acquisition in OocytesWild-type and mutant channels were screened at room temperature for macroscopic Ba 2ϩ currents, 2 to 4 days after RNA injection, using a two-electrode voltage-clamp amplifier (OC-725C, Warner Instruments) as described earlier (20,29,34). Oocytes were routinely injected with 23 nl of a 50 mM EGTA (Sigma) solution 0.5 to 2 h before the experiments.…”

Swapping the I-II intracellular linker between L-type CaV1.2 and R-type CaV2.3 high-voltage gated calcium channels exchanges activation attributes

“…[18][19][20][21] In addition, residues located at the cytoplasmic end of IS6 have been reported to affect channel opening. [22][23][24] These residues seem to coincide with the bundle crossing region where S6 from other domains come together (reviewed by Hering, et al). 6 Thus, it appears coherent that the I-II linker adds a component to the channel gating machinery as it is physically connected to IS6 of the voltage-dependent ion channel family.…”