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

Abstract

We report here the construction of a novel knock-in mouse expressing chimeric α3 nAChR subunits with pharmacological sensitivity to α-bungarotoxin (αBTX). Sensitivity was generated by substituting five amino acids in the loop C (β9-β10) region of the mouse α3 subunit with the corresponding residues from the α1 subunit of the muscle type receptor from Torpedo californica. To demonstrate the utility of the underlying concept, expressed α3[5] subunits were characterized in the superior cervical ganglia (SCG) of homozygous knock-in mice, where the synaptic architecture of postsynaptic α3-containing nAChR clusters could now, for the first time, be directly visualized and interrogated by live-staining with rhodamine-conjugated αBTX. Consistent with the postsynaptic localization of ganglionic nAChRs, the αBTX-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 α3 [5] were abolished by 100 nM αBTX, even in an α7 null background, demonstrating that synaptic throughput in the SCG is completely dependent on the α3-subunit. In addition, we observed that the genetic background of various inbred and outbred mouse lines greatly affects the functional expression of α3[5]-nAChRs, suggesting a powerful new approach for exploring the molecular mechanisms underlying receptor assembly and trafficking. As αBTX-sensitive sequences can be readily introduced into other nicotinic receptor subunits normally insensitive to αBTX, the findings described here should be applicable to many other receptors.

Recent advances in understanding nicotinic receptor signaling mechanisms that regulate drug self-administration behavior

“…Unfortunately, null mutation of the α3 nAChR subunit gene leads to autonomic dysfunction and results in postnatal lethality in mice on a C575BL6 background [145], so examination of α3 knockout mice for nicotine self-administration has been greatly hampered. Interestingly, however, a genetically modified mouse was recently created with pharmacological sensitivity to α-bungaratoxin (α-Bgt) [146]. Specifically, chimeric α3 nAChR subunits were generated by substituting five amino acids in the α3 subunit with the corresponding residues from the muscle α1 subunit from Torpedo californica , rendering α3* nAChRs in these mice sensitive to blockade by α-Bgt [146].…”

Recent advances in gene manipulation and nicotinic acetylcholine receptor biology

“…Caffery and coworkers generated a knock-in mouse line expressing a chimeric α3 nAChR subunit (α3[5] knock-in mice) [57]. Five amino acids in the loop C region of the mouse α3 subunit were substituted with the corresponding residues from the α1 subunit of muscle type nAChR from Torpedo californica.…”

“…This manipulation renders α3* receptors sensitive to the muscle and α7 nAChR antagonist α-BTX [58]. Staining of superior cervical ganglia with rhodamine-conjugated α-BTX as well as pre- and postsynaptic markers showed that α3[5]* receptors colocalized with presynaptic varicosities on the soma and dendrites of sympathetic neurons [57]. α3[5] knock-in mice showed nerve-evoked compound action potentials and excitatory postsynaptic potentials that were smaller than in wild-type mice but blocked by α-BTX.…”

“…In the case of α3[5] mice, the background strain affects viability quite dramatically [57]. Homozygous - but not heterozygous - animals maintained on mixed S129S7/C57BL/6 background die within three months of birth, 50 % of deaths occurring before the third week of life.…”

KV4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxin binding provide a new tool for studies of channel expression and localization

“…Similar methodologies could be used to investigate Kv4.2 interactomes using HAP-tagged Kv4.2. (v) Fluorescent conjugates of Bgtx allow live staining of postsynaptic receptors in sympathetic ganglia from mice engineered with replacement of the native α3 nicotinic receptor subunit with one sensitive to Bgtx 35. Live staining of the wild-type α3 nicotinic receptor subunit in any sympathetic or other neuronal preparation has not been possible with subunit-specific antibodies.…”