The prototoxin LYPD6B modulates heteromeric α3β4-containing nicotinic acetylcholine receptors, but not α7 homomers

Prototoxins are a diverse family of membrane-tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain-containing (LYPD)-6B on alpha 3 beta 4-, alpha 3 alpha 5 beta 4-, and alpha 7-containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human alpha 3, beta 4, and alpha 5 (D and N variants) subunits were expressed in Xenopuslaevis oocytes and tested with or without LYPD6B. We used the 2-electrode voltage-clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist-induced current (I-max), and time constant (tau) of desensitization. For beta 4-alpha 3-alpha 3-beta 4-alpha 3 and beta 4-alpha 3-beta 4-alpha 3-alpha 3, LYPD6B decreased EC50 from 631 to 79 mu M, reduced I-max by at least 59%, and decreased . For beta 4-alpha 3-alpha 5D-beta 4-alpha 3 and beta 4-alpha 3-beta 4-alpha-alpha 5D, LYPD6B decreased I-max by 63 and 32%, respectively. Thus, LYPD6B acted only on (3)(3)(4)(2) and (3)(2)(5D)(4)(2) and did not affect the properties of (alpha 3)(2)(beta 4)(3), alpha 7, or (alpha 3)(2)(alpha 5N)(beta 4)(2) nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (alpha 3)(3)(beta 4)(2) nAChRs to ACh while reducing ACh-induced whole-cell currents. LYPD6B also negatively modulates 34 nAChRs that include the alpha 5D common human variant, but not the N variant associated with nicotine dependence.