Mutations in the M4 domain of Torpedo californica acetylcholine receptor dramatically alter ion channel function

Abstract

Site-directed mutagenesis was used to mutate alpha Cys418 and beta Cys447 in the M4 domain of Torpedo californica acetylcholine receptor expressed in Xenopus laevis oocytes. The M4 region is a transmembrane domain thought to be located at the lipid-protein interface. By whole-cell voltage clamp analysis, mutation of both alpha subunits to alpha Trp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC50 for acetylcholine from 32 microM to 13 microM. Patch measurements of single-channel currents revealed that the alpha Trp418 increased channel open times approximately 28-fold at 13 degrees C with no effect on channel conductance. All of our measured functional changes in the alpha Trp418 mutant are consistent with a simple kinetic model of the acetylcholine receptor in which only the channel closing rate is altered by the mutation. Our results show that changes in protein structure at the putative lipid-protein interface can dramatically affect receptor function.

Tryptophan Substitutions at Lipid-exposed Positions of the Gamma M3 Transmembrane Domain Increase the Macroscopic Ionic Current Response of the Torpedo californica Nicotinic Acetylcholine Receptor

“…Previous findings demonstrated that a single mutation at the postulated protein-lipid surface of the AChR could produce dramatic effects on the closing transition of the channel (Lee et al, 1994;Lasalde et al, 1996;Ortiz-Miranda et al, 1997;Bouzat et al, 1998;Tamamizu et al, 1999Tamamizu et al, , 2000. These results suggest a possible role of the lipid-exposed regions of the AChR on the gating mechanism.…”

“…RNA transcripts were synthesized in vitro as described by Lee et al (1994). The RNA transcripts (10 ng/oocyte at the concentration of 0.2 μg/μl) of α-, β-, γ-, and δ-subunit at a 2:1:1:1 ratio were injected into Xenopus oocytes.…”

“…The expression of nAChR in the oocyte membrane was assayed by measuring the binding of 125 I-α-bungarotoxin (Amersham Life Sciences, Arlington Heights, IL) to intact oocytes according to Lee et al (1994). From 6-35 oocytes per mutation were incubated in 10 nM 125 I-α-bungarotoxin with 5 mg/ml bovine serum albumin in MOR2 at room temperature for 2 hours and the excess toxin was removed by washing 5 times with 1 ml of MOR2.…”

“…To evaluate the contribution of lipid-exposed mutations on multiple subunits to AChR function we co-injected the αC418W mutation (Lee et al, 1994;Ortiz-Miranda, 1997) with γM3 mutations that increase the macroscopic current. Figure 3 shows families of AChinduced currents for the three M3 mutants (F292W, L296W and M299W) coexpressed with the αC418W.…”

Molecular mechanisms of acetylcholine receptor–lipid interactions: from model membranes to human biology

“…Mutations of lipid-exposed residues on M4 alter channel gating showing that there is a direct role for M4 in nAChR function. ( (Li et al 1992;Lee et al 1994;Lasalde et al 1996;Bouzat et al 1998;Tamamizu et al 2000;Mitra et al 2004). As noted, mutation of a lipid facing residue on M4 (αC418W) in the muscle type receptor leads to pathological consequences, demonstrating that M4-lipid interactions are important in Fig.…”