Strychnine activates neuronal α7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains

Recent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric α7 receptors, and that mutating Leu-247 of the α7 nicotinic AcCho receptor-channel domain (L247Tα7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on Xenopus oocytes expressing the chick wild-type α7 or L247Tα7 receptors. In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247Tα(7) receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189–190Sα7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker α-bungarotoxin. Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic α7 receptors. After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1. A pentameric symmetrical model yields the striking conclusion that two identical α7 subunits may be sufficient to determine the functional properties of α7 receptors

Nicotinic acetylcholine receptors assembled from the alpha7 and beta3 subunits

Intracellular recordings were performed in voltageclamped Xenopus oocytes upon injection with a mixture of cDNAs encoding the β3 and mutant α7 (L247Tα7) neuronal nicotinic acetylcholine receptor (nAChR) subunits. The expressed receptors maintained sensitivity to methyllycaconitine and to α-bungarotoxin but exhibited a functional profile strikingly different from that of the homomericL247Tα7 receptor. The heteromericL247T α7 β3 nAChR had a lower apparent affinity and a faster rate of desensitization than L247Tα7 nAChR, exhibited nonlinearity in the I-V relationship, and was inhibited by 5-hydroxytryptamine, much like wild type α7 (WT α7) nAChR. Single channel recordings in cell-attached mode revealed unitary events with a slope conductance of 19 picosiemens ans a lifetime of 5 ms, both values being much smaller than those of the homomeric receptor channel. Upon injection with a mixture of WTα7 and β3 cDNAs, clear evidence was obtained for the plasma membrane assembly of heteromeric nAChRs, although ACh could not activate these receptors. It is concluded that β3, long believed to be an orphan subunit, readily co-assembles with other subunits to form heteromeric receptors, some of which may be negative regulators of cholinergic function.</p

α₅ subunit forms functional α₃β₄α₅ nAChRs in transfected human cells

NAChRs heterologously expressid in human cells after transient transfection with α₃β₄α₅ or α₃β₄ subunit cDNAs exhibited similar sensitivities to antogonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of α₅ α₃ and β₄ subunits. One component was comparable to that obtained in α₃β₄-transfected cells, while the additional component, putatively attributed to an α₃β₄α₅ nAChR population, showed a Hill coeeficient &gt; 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the α₅ subunit participates in the assembly of α₃β₄α₅ nAChRs compelxes in human cells, adding a new member to the family of neuronal nicotinic receptors