FUNCTIONAL ROLE OF THE M3 TRANSMEMBRANE DOMAIN OF THE NICOTINIC ACETYLCHOLINE RECEPTOR: MUTATIONS AT POSITION 8`ALTER ION CHANNEL KINETICS.

DE ROSA, MARIA J.; RAYES, DIEGO; SPITZMAUL, GUILLERMO; BOUZAT, CECILIA

Buenos Aires

Congreso; 18 th Biennial Meeting of the International Society for Neurochemistry (ISN); 32nd Annual Meeting of the American Society for Neurochemistry (ASN); 2001

International Society for Neurochemistry (ISN); American Society for Neurochemistry (ASN)

The nicotinic receptor (AChR) is a pentamer of homologous subunits with composition a2bde in adult muscle. Each subunit contains four transmembrane domain (M1-M4). How M3 domain contributes to channel gating still remains unknown. Position 8`of the M3 domain is phenylalanine in all heteromeric a subunit, but is a nonaromatic residue in homomer-forming a subunits and nona subunits. Given this particular pattern of conservation, we study its contribution to channel gating by combining site-directed mutagenesis and single channel recordings. Replacement of phenylalanine in 8` of aM3 by an non-aromatic residue increases the mean open time of the resulting AChRs. In contrast, substitution of leucine by phenylalanine at the homologous position of b, d and e subunits decreases the mean open time, the b subunit being the most sensitive to mutation. Studies combining wild-type a subunits with a different number of mutant non-a subunits reveal that the mean open time decreases as a function of the number of phenylalanines at 8`M3. The plot of the ratios of mean open time values for each mutant AChR as a function of the total number of phenylalanine residues in 8`shows that: (i) the mean open time depends on the number of phenylalanine residues; (ii) all subunits contribute independently and slightly asymmetrically to channel closing; and (iii) each phenylalanine decreases the energy barrier of the closing process by ~0.4 kcal/mol. Thus by dissecting the effects of mutations on the closing rate constant we show that M3 of all subunits contribute to channel activation and that the presence of phenylalanines in 8`increases the rate of channel closing.