Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes

Abstract

This report analyzes the contribution of individual nicotinic acetylcholine receptor (AChR) subunits to the single-channel properties of the AChR ion channel. By in vitro synthesis of mRNA from cDNA clones encoding each AChR subunit (alpha, beta, gamma, and delta) from mouse BC3H-1 cells and Torpedo electric organ and microinjection of appropriate mRNA combinations into Xenopus oocytes, we studied the single-channel properties of both 'homologous' (all subunits from the same species) and 'hybrid' (subunits from both species) AChRs as they were expressed in the oocyte membrane. AChR expression was determined by surface binding of 125I-labeled alpha-bungarotoxin to intact oocytes, and those with binding sites of 1 fmol/cell or more were chosen for patch-clamp studies. Our results indicate the following: (1) Species difference in single-channel conductance can be explained largely by the charge distribution flanking the M2 transmembrane domain. (2) The alpha and delta subunits from mouse AChR independently lengthen the channel open time, in some cases by 10-fold; the beta subunit from mouse shortens the channel open time; the mouse gamma subunit lengthens open time less dramatically. (3) Voltage sensitivity, as measured by the ratio of channel open times at -60 mV and +60 mV, is influenced by the beta and delta subunits, in agreement with our previous study by two-electrode voltage-clamp recording. We conclude that single-channel properties of the AChR are governed by multiple elements located on different AChR subunits.