Engineering neuronal nicotinic acetylcholine receptors with functional sensitivity to α-bungarotoxin: a novel α3-knock-in mouse

We report here the construction of a novel knock-in mouse expressing chimeric alpha 3 nicotinic acetylcholine receptor (nAChR) subunits with pharmacological sensitivity to alpha-bungarotoxin (alpha BTX). Sensitivity was generated by substituting five amino acids in the loop C (beta 9-beta 10) region of the mouse alpha 3 subunit with the corresponding residues from the alpha 1 subunit of the muscle type receptor from Torpedo californica. To demonstrate the utility of the underlying concept, expressed alpha 3[5] subunits were characterized in the superior cervical ganglia (SCG) of homozygous knock-in mice, where the synaptic architecture of postsynaptic alpha 3-containing nAChR clusters could now, for the first time, be directly visualized and interrogated by live-staining with rhodamine-conjugated alpha BTX. Consistent with the postsynaptic localization of ganglionic nAChRs, the alpha BTX-labeled puncta colocalized with a marker for synaptic varicosities. Following in vivo deafferentation, these puncta persisted but with significant changes in intensity and distribution that varied with the length of the recovery period. Compound action potentials and excitatory postsynaptic potentials recorded from SCG of mice homozygous for alpha 3[5] were abolished by 100 nm alpha BTX, even in an alpha 7 null background, demonstrating that synaptic throughput in the SCG is completely dependent on the alpha 3-subunit. In addition, we observed that the genetic background of various inbred and outbred mouse lines greatly affects the functional expression of alpha 3[5]-nAChRs, suggesting a powerful new approach for exploring the molecular mechanisms underlying receptor assembly and trafficking. As alpha BTX-sensitive sequences can be readily introduced into other nicotinic receptor subunits normally insensitive to alpha BTX, the findings described here should be applicable to many other receptors.