MOLECULAR PORE STRUCTURE OF VOLTAGE-GATED SODIUM AND CALCIUM CHANNELS

1. Voltage-gated ion channels have a high homology in their primary st ructure but yet show quite distinct ion selection properties. Therefor e, ion selection was studied in voltage-gated wild-type and mutant sod ium (Na RBII) and calcium (Ca BI) channels, which were heterologously expressed in Xenopus oocytes, using two-electrode voltage clamp and pa tch clamp methods. 2. In order to facilitate the electrophysiological experiments, fast sodium channel inactivation was abolished by introdu cing the point mutation F1489Q into the linker between the homologous repeats III and IV. 3. The ion pore in rat brain sodium channel II was located by testing single-point mutants for their sensitivity to exte rnally applied tetrodotoxin (TTX), leading to a model in which part of the linker S5-S6 folds into the membrane to form at least part of the ion pore; two amino acids from each domain are thereby of major impor tance for TTX association and ion permeation, presumably forming two r ings of predominantly charged residues in three-dimensional space (out er ring: E387, E945, M1425, D1717; inner ring: D384, E942, K1422, A171 4). 4. When the residue F385 in repeat I of Na RBII channel is mutated to a cysteine, the residue being at the homologous position in cardia c sodium channels, the channel lost its high sensitivity for TTX and g ained a high sensitivity for external Cd2+ and Zn2+, properties found in cardiac sodium channels. Mutation of the other cysteine in the pore region, C940, had no effect on channel block by TTX or by external di valent cations. 5. The inner ring in sodium channels constitutes a sel ectivity filter: after mutation of residues K1422 and A1714 into gluta mates, the homologous residues in calcium channels, the sodium channel s show ion selection properties similar to calcium channels: a) no sel ectivity among monovalent cations, b) strong block of monovalent curre nt by divalent cations, c) permeation of divalent cations. 6. Reverse mutations in the BI calcium channel, where glutamate residues were mut ated into neutral glutamines, showed that also in this channel these a mino acids form a selectivity filter. The individual residues interact differently with the permeating ions suggesting an asymmetric spatial arrangement of the four pore-forming regions of the ion channel pen.