TY - JOUR
T1 - Molecular motions of the outer ring of charge of the sodium channel
T2 - Do they couple to slow inactivation?
AU - Xiong, Wei
AU - Li, Ronald A.
AU - Tian, Yanli
AU - Tomaselli, Gordon F.
PY - 2003/9/1
Y1 - 2003/9/1
N2 - In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain β 1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C+E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the α carbons of these residues of no more than 7 Å. The redox catalyst Cu(II) (1,10-phenanthroline) 3 (Cu(phe)3) reduced the peak current of double mutants (E403C+E758C, E403C+D1241C, E403C+D1532C, and D1241C+D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe)3 modification of outer charged ring paired mutants E403C+E758C, E403C+D1241C, and E403C+D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe)3 modification of other double mutants such as E403C+W756C, E403C+W1239C, and E403C+W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge.
AB - In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain β 1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C+E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the α carbons of these residues of no more than 7 Å. The redox catalyst Cu(II) (1,10-phenanthroline) 3 (Cu(phe)3) reduced the peak current of double mutants (E403C+E758C, E403C+D1241C, E403C+D1532C, and D1241C+D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe)3 modification of outer charged ring paired mutants E403C+E758C, E403C+D1241C, and E403C+D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe)3 modification of other double mutants such as E403C+W756C, E403C+W1239C, and E403C+W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge.
KW - Cysteine mutagenesis
KW - Disulfide bond
KW - Electrophysiology
KW - rNav1.4 channel
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U2 - 10.1085/jgp.200308881
DO - 10.1085/jgp.200308881
M3 - Article
C2 - 12913092
AN - SCOPUS:0141513676
SN - 0022-1295
VL - 122
SP - 323
EP - 332
JO - Journal of General Physiology
JF - Journal of General Physiology
IS - 3
ER -