MOUSE-TORPEDO CHIMERIC ALPHA-SUBUNIT USED TO PROBE CHANNEL-GATING DETERMINANTS ON THE NICOTINIC ACETYLCHOLINE-RECEPTOR PRIMARY SEQUENCE

1. To determine if structural domains are important for nicotinic acet ylcholine receptor (nAChr) channel function, six mouse-Torpedo chimeri c alpha-subunits were constructed (Fig. 2) and coexpressed with Torped o californica beta-, gamma-, and delta-subunits in Xenopus laevis oocy tes. 2. nAChRs containing a chimeric alpha-subunit were examined by vo ltage- and patch-clamp methods to determine their functional character istics, Dose-response curves from voltage-damped oocytes were used to estimate EC(50)'s and Hill coefficients. Whole-cell currents were norm alized against the alpha-bungarotoxin (alpha-BTX) binding sites to obt ain normalized responses to acetylcholine (ACh). Open time constants a t 4 mu M ACh were used to examine single-channel behavior. 3, The EC(5 0) for ACh was modulated by the N-terminal half of the alpha-subunit. When the Torpedo subunit sequence between position 1 and position 268 was replaced by mouse sequence, the EC(50) Shifted toward the value fo r the wild-type mouse subunit. Replacement of either the 1-159 or the 160-268 positions of the Torpedo sequence with the mouse sequence lowe red the EC(50). This suggests that at least two regions play a role in determining the EC(50). 4. When the primary sequence (160-268) of the Torpedo alpha-subunit was introduced in the mouse alpha-subunit (Tl60 -268), the expressed chimeric receptor was nonfunctional. The inverse chimera (M160-268) was functional and the open time constant and EC(50 ) were similar to those of mouse but the normalized response was chara cteristic of Torpedo. 5. The normalized macroscopic response to ACh (3 00 mu M) Of the chimera containing the mouse alpha-subunit showed a ni nefold increase relative to the Torpedo wild type. Receptors which con tain the C terminal of the mouse alpha-subunit also show an increase i n the maximum normalized current. Receptors with the alpha-subunit whi ch contain the Torpedo C-terminal sequence have a lower normalized res ponse. 6. The combined results suggest that AChR channel function is m odulated by structural determinants within the primary sequence. These structural domains might modulate channel function through specific a llosteric interactions. The lack of response of the T160-268 chimera s uggests that a critical interaction essential for the coupling of agon ist binding and channel gating was disrupted. This result suggests tha t the interaction of structural domains within the nAChR primary struc ture are essential for channel function and that these intractions cou ld be very specific within different nAChR species.