Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region.
Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.
Cohen
B N
BN
Division of Biology 156-29, California Institute of Technology, Pasadena 91125.
Labarca
C
C
Czyzyk
L
L
Davidson
N
N
Lester
H A
HA
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology*; D004247:DNA / Q000032:analysis / Q000235:genetics; D008433:Mathematics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics / Q000502:physiology*; D009865:Oocytes / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D011978:Receptors, Nicotinic / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D012964:Sodium / Q000493:pharmacokinetics*; D014101:Torpedo; D014325:Tromethamine / Q000493:pharmacokinetics*; D014981:Xenopus
D011978:Receptors, Nicotinic; D014325:Tromethamine; D004247:DNA; D012964:Sodium