GLUTAMATE SUBSTITUTION IN REPEAT-IV ALTERS DIVALENT AND MONOVALENT CATION PERMEATION IN THE HEART CA2+ CHANNEL

In voltage-gated ion channels, residues responsible for ion selectivit y were identified in the pore-lining SS1-SS2 segments, Negatively char ged glutamate residues (E393, E736, E1145, and E1446) found in each of the four repeats of the alpha(1C) subunit were identified as the majo r determinant of selectivity in Ca2+ channels. Neutralization of gluta mate residues by glutamine in repeat I (E393Q), repeat III (E1145Q), a nd repeat IV (E1446Q) decreased the channel affinity for calcium ions 10-fold from the wild-type channel. In contrast, neutralization of glu tamate residues in repeat II failed to significantly alter Ca2+ affini ty. Likewise, mutation of neighboring residues in E1149K and D1450N di d not affect the channel affinity, further supporting the unique role of glutamate residues E1145 in repeat III and E1446 in repeat IV in de termining Ca2+ selectivity. Conservative mutations E1145D and E1446D p reserved high-affinity Ca2+ binding, which suggests that the interacti on between Ca2+ and the pore ligand sites is predominantly electrostat ic and involves charge neutralization. Mutational analysis of E1446 sh owed additionally that polar residues could achieve higher Ca2+ affini ty than small hydrophobic residues could. The role of high-affinity ca lcium binding sites in channel permeation was investigated at the sing le-channel level. Neutralization of glutamate residue in repeats I, II , and III did not affect single-channel properties measured with 115 m M BaCl2. However, mutation of the high-affinity binding site E1446 was found to significantly affect the single-channel conductance for Ba2 and Li+, providing strong evidence that E1446 is located in the narro w region of the channel outer mouth. Side-chain substitutions at 1446 in repeat IV were used to probe the nature of divalent cation-ligand i nteraction and monovalent cation-ligand interaction in the calcium cha nnel pore. Monovalent permeation was found to be inversely proportiona l to the volume of the side chain at position 1446, with small neutral residues such as alanine and glycine producing higher Li+ currents th an the wild-type channel. This suggests that steric hindrance is a maj or determinant for monovalent cation conductance. Divalent permeation was more complex. Ba2+ single-channel conductance decreased when small neutral residues such as glycine were replaced by bulkier ones such a s glutamine. However, negatively charged amino acids produced single-c hannel conductance higher than predicted from the size of their side c hain. Hence, negatively charged residues at position 1446 in repeat IV are required for divalent cation permeation.