Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels

Abstract

Voltage-gated Ca2+ channels link changes in membrane potential to the delivery of Ca2+, a key second messenger for many cellular responses. Ca2+ channels show selectivity for Ca2+ over more plentiful ions such as Na+ or K+ by virtue of their high-affinity binding of Ca2+ within the pore. It has been suggested that this binding involves four conserved glutamate residues in equivalent positions in the putative pore-lining regions of repeats I-IV in the Ca2+ channel a1 subunit. We have carried out a systematic series of single amino-acid substitutions in each of these positions and find that all four glutamates participate in high-affinity binding of Ca2+ or Cd2+. Each glutamate carboxylate makes a distinct contribution to ion binding, with the carboxylate in repeat III having the strongest effect. Some single glutamate-to-lysine mutations completely abolish micromolar Ca2+ block, indicating that the pore does not possess any high-affinity binding site that acts independently of the four glutamate residues. The prevailing model of Ca2+ permeation must thus be modified to allow binding of two Ca2+ ions in close proximity, within the sphere of influence of the four glutamates. The functional inequality of the glutamates may be advantageous in allowing simultaneous interactions with multiple Ca2+ ions moving single-file within the pore. Competition among Ca2+ ions for individual glutamates, together with repulsive ion-ion electrostatic interaction, may help achieve rapid flux rates through the channel.

Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development

“…The inability of the Ca V 1.2 4EQ channel to rescue the Ca V 1.2 morpholino effects ( Figure 3, A and C) and the smaller jaws observed after pharmacologic blockade of Ca V 1.2 with nisoldipine ( Figure 3, D and E) provide additional evidence that mandibular development depends directly on Ca 2+ influx through Ca V 1.2. Dihydropyridines have not been shown to disrupt the Ca V 1.2 macromolecular complex, and the Ca V 1.2 4EQ channel remains a voltage-sensitive cation channel -though one that is not able to permeate Ca 2+ due to loss of the high-affinity binding site in the pore (11). Thus, we conclude that functions other than Ca 2+ permeation are unlikely to contribute to the Ca V 1.2-dependent regulation of mandibular development.…”

“…We next tested whether jaw development depended upon Ca 2+ influx through Ca V 1.2 with a Ca 2+ -impermeant rabbit Ca V 1.2, in which 4 key aspartate residues in the channel's poreforming α 1C subunit were mutated to glutamine. This 4EQ mutant (Ca V 1.2 4EQ ) retains its ability to scaffold other signaling proteins (e.g., auxiliary subunits) and to conduct ions, but completely blocks Ca 2+ permeation (11). Unlike Ca V 1.2 WT or Ca V 1.2 TS , Ca V 1.2 4EQ did not restore mandible size when coinjected with the Ca V 1.2 knockdown morpholino cocktail ( Figure 3, A and C).…”

Endogenous cardiac Ca2+ channels do not overcome the E‐C coupling defect in immortalized dysgenic muscle cells: evidence for a missing link

“…Consequently, part of the topological arrangement of the cq in motif IV is altered. The translation stops in the mutant mdg oq after the motif IV SS 1 region and the critical SS2 segment that carries glutamic acid residues important for Ca 2+ selectivity are missing [24][25][26]. Further, the IVS 6 and the C-terminal tail are deleted (Fig.…”

“…Second, in the mutant protein part of the IVSS 1, the IVSS2, the extracellular connecting loop to IVS6, the IVS 6 and the entire C-terminal cytoplasmic tail is missing. Some of these segments, in particular the IVSS1 and IVSS2, are well-identified integral components of the high-affinity Ca 2+ filtration units [24][25][26]; the putative E-F hand [27] on the Cterminal tail may also be involved in Ca 2+ binding. Based on hydropathy analysis and prediction of secondary structure, we suggest that the C-terminus of the mutant protein will be largely globular and localized intracellularly.…”

Identifying the elusive link between amino acid sequence and charge selectivity in pentameric ligand-gated ion channels

“…In K + -selective channels (31), voltage-dependent Na + (32) and Ca 2+ channels (33), cyclic-nucleotide-gated channels (34), and ClC Cl -channels (35), for example, the identity of these chemical groups has been firmly established on the basis of electrophysiological and/or X-ray crystallographical observations. In pLGICs, in GLIC (PDB code 4HFI) GLIC (PDB code 4HFI) α1 GluCl (PDB code 3RHW) 1 GluCl (PDB code 3RHW) contrast, although the sequence conservation and pore location of the oppositely charged side chains at positions -1′ and 0′ may have suggested that they are the main contributors to charge selectivity, the experimental data recorded before this work had failed to provide compelling evidence for this idea.…”