CONTRIBUTION OF THE M1 TRANSMEMBRANE DOMAIN OF THE MUSCLE NICOTINIC RECEPTOR TO CHANNEL GATING

SPITZMAUL, GUILLERMO; BOUZAT, CECILIA

Buenos Aires

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

International Society for Neurochemistry

The nicotinic receptor (AChR) is a pentamer of homologous subunits with composition Alpha2BetaEpsilonDelta in adult muscle. Each subunit contains four transmembrane domains (M1-M4). The first transmembrane domain (M1) has been shown to be associated with both the lipid bilayer and the ion pore. However, its functional role still remains unclear. To gain new insights into the contribution of M1 to channel gating we combine site-directed mutagenesis with AChR expression in HEK cells and single-channel recordings. Position 15? of the M1 domain is phenylalanine (F) in all a subunits which form heteromeric AChRs but is isoleucine (I) in non-a and homomeric a-subunits. Given the peculiar pattern of conservation of this position we here explore its contribution to channel gating. AChRs containing the mutant aF15?I and eI15?F subunits show increased open durations with respect to wild-type AChRs, being their mean open times 2-fold longer than that of the control.  Replacing isoleucine by phenylalanine in the b subunit (bI15?F) leads to greater changes in the duration of the open state. Open time histograms show a main component whose duration is 7-fold longer than that of wild-type AChRs.  In contrast, AChRs channels containing the mutant dI15?F subunit are kinetically similar to wild-type AChRs. In summary, we here show that the M1 domain contributes to channel gating and that position 15? governs mainly the rate of channel closing. In addition, our results reveal that the different subunits forming the pentameric receptor make asymmetric contributions to channel gating.