Modulation of nicotinic acetylcholine receptors by strychnine

Abstract

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (alpha1beta1gammadelta, alpha1beta1gamma, and alpha1beta1delta) and neuronal (alpha2beta2 and alpha2beta4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 microM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was alpha1beta1gammadelta > alpha2beta4 > alpha2beta2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of approximately 0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex.

Figure 1

Block of AcCho-current by strychnine. (A) Control current evoked by AcCho in an oocyte expressing neuronal α₂β₄ AcChoRs. After 3 min, the oocyte was superfused with strychnine alone, and then the AcCho-current was elicited again in the continuous presence of strychnine. (B) Superimposed records of AcCho-current showing the effects of 1 nM and 10 μM strychnine. (C) Control current, then simultaneous application of AcCho plus strychnine, and the recovered AcCho-current. The records were obtained from the same oocyte. For this and subsequent figures, the membrane was voltage-clamped at −60 mV, and the timings of drug applications are indicated by continuous bars for AcCho and dashed bars for strychnine above the records and by brief depolarizing pulses used to monitor membrane conductance changes. Inward currents are denoted by downward deflections of the trace.

Figure 2

Block of muscle and neuronal AcChoRs by strychnine. The first AcCho-current in each trace of Inset corresponds to the control current elicited by AcCho on muscle α₁β₁γδ, neuronal α₂β₄, or α₂β₂ AcChoRs. The second AcCho-current was evoked by simultaneous superfusion of AcCho plus strychnine. The third response is the recovered AcCho-current recorded 6 min after withdrawing the drugs. The plot corresponds to AcCho-current recovery from block by 200 μM strychnine. Data were normalized to the control AcCho-current before strychnine application. Strychnine was applied before (filled circles) or together with (open circles) AcCho. Data were obtained from the same oocyte expressing muscle α₁β₁γδ AcChoRs. The continuous lines are fittings to single exponential functions.

Figure 3

(A) Sample dose-response relationships for strychnine block of muscle α₁β₁γδ (circles), neuronal α₂β₄ (triangles), and neuronal α₂β₂ (diamonds) AcChoRs. The currents were elicited by 2 μM AcCho for α₁β₁γδ and 50 μM AcCho for α₂β₄ and α₂β₂ receptors. The continuous lines represent least squares fit to the relation I_[strychnine] = I₀·IC₅₀/([strychnine]^nH + IC₅₀^nH), where I_[strychnine] is the AcCho-current amplitude inhibited by strychnine, I₀ is the control peak current, IC₅₀ is the half-inhibitory concentration of strychnine, and nH is the Hill coefficient. (B) Amplitude of progressive control AcCho-current (open diamonds) and AcCho-current in the presence of the strychnine concentrations shown in A for the α₂β₂ receptors (filled diamonds).

Figure 4

Strychnine block of muscle AcChoRs is noncompetitive. Dose-response curve for AcCho-current in the absence (filled circle) and presence (open circles) of strychnine measured in the same oocyte. Continuous lines represent least squares fits to the Hill equation I_[AcCho] = I_max·[AcCho]ⁿ/([AcCho]^nH + EC₅₀^nH), where I_[AcCho] is the AcCho dose-dependent current amplitude, I_max is the amplitude of the control AcCho-current, EC₅₀ is the half-excitatory concentration of AcCho, and nH is the Hill coefficient. (Inset) Membrane currents elicited by two concentrations of AcCho superfused on one oocyte expressing muscle α₁β₁γδ AcChoRs. After 4 min, AcCho and 10 μM strychnine were coapplied.

Figure 5

Effects of strychnine as a function of membrane potential. I–V relationships are shown in the absence and presence of strychnine for muscle α₁β₁γδ (A), neuronal α₂β₂ (B), and neuronal α₂β₄ (C) AcChoRs. The oocytes were maintained at a potential of −60 mV, and brief voltage steps were applied from −120 to 40 mV.

Figure 6

Effects of strychnine on AcChoRs of different subunit composition. The bars indicate the fraction of the AcCho-current remaining at the end of a 2-min application of 10 μM strychnine. (Inset) Left traces are control current evoked by AcCho on an oocyte expressing α₁β₁γ receptors and a superimposed record in which strychnine was coapplied with AcCho. The right trace shows the current evoked by AcCho on an oocyte expressing α₁β₁δ AcChoRs and inhibited by strychnine.

Figure 7

(A) AcCho-current mediated by α₁β₁δ AcChoRs. The oocyte was held at a membrane potential of −60 mV, with 20 mV steps from −140 to 40 mV, in normal Ringer solution during AcCho application and during coapplication with 10 μM strychnine. (B) Comparison between the current elicited by AcCho alone and that in the presence of 10 μM strychnine, in a logarithmic scale, as a function of membrane potential, on oocytes expressing α₁β₁γδ (filled squares), α₁β₁γ (open circles), or α₁β₁δ (filled circles) AcChoRs. The continuous lines are the fits to one-site blocking model (see text).