Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations.

Abstract

We measured the permeability ratios (Px/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xen0pus oocytes for alkali metal and organic cations using shifts in the biionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (t~Thr244, 13Gly255, ~/Thr253, 8Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced Pc~/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na + and glycine methyl ester/Na + permeability ratios. Two subunit alterations also affected selectivity: omission of the 8 subunit reduced PcJPsa by 16%, and substitution ofXenopus 8 for mouse 8 increased P~nidi~ium/PNa more than twofold and reduced Pcs/PN~ by 34% and PLi/PN~ by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 ,~; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/ PNa by 38% and Pbutylanunonitun/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222.

Nicotinic Acetylcholine Receptors

“…The threonine residue at the point of maximum constriction (Torpedo αT244), when substituted by other residues of different volume, has a pronounced effect on ion flow, as if it were at the narrowest part of the open pore (Villarroel & Sakmann, 1992). The diameter of this most constricted portion of the channel, based on permeability measurements made with small uncharged molecules of different size, is about 10Å (Dwyer et al, 1980;Cohen et al, 1992). This value is similar that indicated by the structural results.…”

Novel Seizure Phenotype and Sleep Disruptions in Knock-In Mice with Hypersensitive α4*Nicotinic Receptors

“…Previous studies show that mutations in the M2 region can alter the ion selectivity of nicotinic receptors, although mutations at the 2Ј position appear to have the greatest effect (Cohen et al, 1992;Wang and Imoto, 1992). To determine whether the L9ЈA mutation altered ion selectivity of ␣4␤2 receptors for three physiologically relevant cations (Na ϩ , K ϩ , and Ca 2ϩ ), we substituted K ϩ or Ca 2ϩ for extracellular Na ϩ and measured the shift in reversal potential (E r ) of the ACh response (see Materials and Methods).…”

The structural basis of function in Cys-loop receptors

“…Five symmetrically placed M2 helices from each of the five subunits create a hydrophobic region that is 3 Å at its narrowest and less than 3Á5 Å over a distance of approximately 8 Å in the closed state, and has been referred to as a hydrophobic girdle (Miyazawa et al 2003). Ion permeabilities suggest that the diameter of the open channel is between 7Á4 Å and 8Á4 Å for cation channels and between 5Á2 Å and 6Á2 Å for anion channels (Brown et al 1998 ;Cohen et al 1992 ;Fatima-Shad & Barry, 1993 ;Rundstrom et al 1994 ;Wang & Imoto, 1992). Originally, the channel gate was predicted to be close to the cytoplasmic end of M2 (Wilson & Karlin, 1998 ;Wilson et al 2000).…”