Strychnine activates neuronal alpha7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains

Abstract

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 alpha7 receptors, and that mutating Leu-247 of the alpha7 nicotinic AcCho receptor-channel domain (L247Talpha7; 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 alpha7 or L247Talpha7 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 L247Talpha(7) receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189-190Salpha7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker alpha-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 alpha7 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 alpha7 subunits may be sufficient to determine the functional properties of alpha7 receptors.

Figure 1

Effects of strychnine on AcCho-induced currents. (A) Strychnine dose/AcCho current response relations in oocytes expressing WTα7 (■) or mut1 receptors (□). Peak currents, evoked by AcCho (100 μM in ■; 0.3 μM in □) coapplied with strychnine and normalized to the response to AcCho alone. Data represent mean ± SEM (6/3 in ■; 7/2 in □). Data without bars, mean of two oocytes. (Inset, Left) Sample superimposed I_AcCho elicited by AcCho at indicated concentrations (in μM), alone (∗), or together with strychnine (0.3 μM) in an oocyte expressing mut1 receptor. (Inset, Right) Sample I_AcCho elicited by 100 μM alone (horizontal solid bar) or together with strychnine (1 μM; middle trace) in an oocyte expressing WTα7. (B) Current-voltage relation from one oocyte held at various potentials: (●) 100 μM AcCho and (○) 100 μM AcCho coapplied with 1 μM strychnine (representative of four oocytes, one donor). AcCho was applied at 3-min intervals. Solid lines represent second-order polynomial fits to the data. (C) Current-voltage relations in one oocyte exposed to AcCho alone or AcCho plus strychnine (0.3 μM). Representative of five oocytes, two donors.

Figure 2

Effects of AcCho and strychnine on oocytes expressing mut2 receptors. AcCho dose/I_AcCho relationship (●) and strychnine dose/I_strych relationship (○) best-fitted to a single Hill equation. Peak I_AcCho and I_strych were normalized to that evoked by 3 mM AcCho (I_AcCho mean amplitude: −2.8 μA) or by 50 μM strychnine (I_strych mean amplitude: −1.5 μA), respectively. Each point is the mean ± SEM (●, 12/3; ○, 24/8). Note expected decrease of I_strych values at high strychnine concentrations, probably because of a channel block mechanism. (Inset, Right) Sample records of I_AcCho in two oocytes: (Upper) oocyte held at −60 mV; (Lower) another oocyte held at −100 mV. (Inset, Left) Sample records of I_strych in two other oocytes, held as at right. Note absence of shoulder in the I_AcCho elicited by 1 mM AcCho at right and its presence in I_strych (at −60 mV), and wash kick with long application of 0.5 mM strychnine at −100 mV.

Figure 3

I–V relationships in oocytes expressing mut2 receptors. (A) Voltage ramps (−130 to 30 mV membrane potential; 1.8-s duration) in the presence of either AcCho (35 μM) or strychnine (5 μM) in the same oocyte. (Inset) Sample records of strychnine currents from another oocyte tested with voltage steps (from bottom to top: −100 mV, −60 mV, −40 mV, +30 mV, inward current, downward). (B) I–V relationships performed with voltage steps showing linearity for I_AcCho and rectification at hyperpolarized potential for I_strych. AcCho concentration, 1 mM; strychnine, 500 μM.

Figure 4

Properties of mut2 channels activated by AcCho (Left) or strychnine (Right), in one oocyte injected with mut2 cDNA. (A) Macroscopic voltage-clamped currents. Holding potential, −100 mV. (B) Single-channel currents elicited by AcCho and strychnine in one outside-out patch. Dashed lines indicate amplitude levels of elementary inward currents (downward deflections). Holding potential, −50 mV. (C) Distributions of single-channel amplitudes, detected from recordings shown in B and best-fitted by the sum (thick line) of two Gaussian functions. Unitary conductances as indicated.

Figure 5

Membrane currents from oocytes injected with a mixture of cDNA encoding mut1 and mut2 subunits with ratio of 1:1. AcCho dose/I_AcCho relationship (●) and strychnine dose/I_strych relationship (○) both best-fitted to a single Hill equation from the same oocytes. For AcCho the EC₅₀ and n_H values determined from the fit were 0.77 μM and 1.9, respectively. The AcCho currents were normalized to that evoked by 10 μM AcCho (I_AcCho mean amplitude: −2.4 μA). For the same oocytes strychnine EC₅₀ and n_H values were 1.3 μM and 1.4, respectively. Strychnine currents were normalized to that evoked by 50 μM strychnine (I_strych mean amplitude: −0.92 μA). Each point represents the mean ± SEM (10/2). Dashed line represents the theoretical curve for the expression of mut1 and mut2 receptors and lack of hybrids (values of EC₅₀ and n_H for pure mut1 and mut2 receptors, as in Table 1). (Insets) Sample I_AcCho (Left) and I_strych (Right) at indicated concentrations.

Figure 6

Diagram illustrating the pentameric symmetrical model and predicted hybrid nAcChoR diversity. Numbers represent fractions of the eight possible combinations determined by the binomial equation assuming equal efficiency in the expression and assembly of mut1 or mut2 subunits. (Upper Numbers) cDNA injection ratio 1:1; (Lower Numbers) ratio 1:5. The binomial equation used to calculate the above fractions was: p^rq^n-r × n!/[r!(n − r)!], where p and q are the incorporation probabilities of mut1 or mut2 subunits, and n is the number of subunits in the receptor complex (five) taken r subunits at a time. Note higher probability (about 60%) for combinations two vs. three subunits in the ratio 1:1.

Figure 7

AcCho dose/I_AcCho response relationship best-fitted to a sum of two Hill equations in oocytes injected with a mixture of cDNA encoding mut1 and mut2 subunits with ratio of 1:5. The EC₅₀, n_H, and weight values determined from the fit were: 1 μM, 2, and 55%, respectively (first component), and 25 μM, 2.5 and 45%, respectively (second component). The peak AcCho currents were normalized to that evoked by 100 μM AcCho (I_AcCho mean amplitude: −1.4 μA). Each point represents the mean ± SEM (6/2). A single Hill equation fit was statistically less significant (F-distribution test). (Inset) Sample I_AcCho.