Modulation of nicotinic acetylcholine receptors by strychnine

Abstract

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (␣ 1 ␤ 1 ␥␦, ␣ 1 ␤ 1 ␥, and ␣ 1 ␤ 1 ␦) and neuronal (␣ 2 ␤ 2 and ␣ 2 ␤ 4 ) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 M) 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 ␣ 1 ␤ 1 ␥␦ > ␣ 2 ␤ 4 > ␣ 2 ␤ 2 . 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 voltageindependent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltagedependent, with an electrical distance of Ϸ0.35. 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 receptorchannel complex.Nicotinic acetylcholine receptors (AcChoRs) are members of a gene superfamily that includes GABA A , glycine, and 5HT 3 receptors (1). They are activated by the neurotransmitter acetylcholine (AcCho), and they mediate fast synaptic transmission at the neuromuscular junction and throughout the vertebrate nervous system (2-4). Additionally, AcChoRs are regulated by a wide variety of substances (5, 6), including strychnine, a selective antagonist of glycine-gated Cl Ϫ channels (7) that inhibits AcChoRs at the neuromuscular junction (8) and different types of neurons (9-13). Moreover, it appears that this inhibition depends on the subtype of nicotinic receptor involved. For instance, in bovine adrenal chromaffin cells and rat hippocampal neurons, strychnine inhibits AcChoRs competitively whereas for ␣ 4 ␤ 2 -containing AcChoRs the inhibition is noncompetitive (10, 13). Here, we report the effects of strychnine on muscle AcChoRs made up of ␣ 1 ␤ 1 ␥␦, ␣ 1 ␤ 1 ␥, or ␣ 1 ␤ 1 ␦ muscle subunits and on two subtypes of neuronal AcChoRs (␣ 2 ␤ 2 or ␣ 2 ␤ 4 ). MATERIALS AND METHODSThe methods were as previously described (14-16). In brief, cDNA clones encoding embryonic mouse muscle AcChoR subunits (␣ 1 , ␤ 1 , ␥, and ␦) or rat neuronal AcChoR subunits (␣ 2 , ␤ 2 , or ␤ 4 ) were used to make cRNAs that were suspended in RNase-free water at a concentration of 1 g͞l. Mixtures then were made with equal quantities of the requ...

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

“…We report also that strychnine does not change the desensitization or voltage-dependence of WT␣7 or mut1 receptors, but acts on them as a ''pure'' competitive blocker. Nonetheless, in some heteromeric neuronal nAcChoRs, the strychnine block is noncompetitive and voltage dependent (1). Thus, our findings taken together with previous data (1, 2, 4-7) suggest avenues for the design of novel therapeutic agents for neurodegenerative diseases such as Alzheimer's and Parkinson's, at which the ␣7 subunit appears to play a role in both disease genesis and progression (15).…”

Quercetin Inhibits α3β4 Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed inXenopusOocytes

“…On the other hand, the previous reports have shown that various agents such as serotonin, strychnine, Ca 2＋ channel blockers, polyamines, steroids such as progesterone and hydrocortisone, ethanol, and metal ion like Zn 2＋ , regulate muscle or neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes [28][29][30][31][32][33][34][35]. Interestingly, the mechanism (i.e., voltage dependence or competition with acetylcholine for binding site) by which these substances regulate nicotinic acetylcholine receptors depend on the receptor subunit composition.…”

A Hybrid Electrical/Chemical Circuit in the Spinal Cord Generates a Transient Embryonic Motor Behavior

“…Zebrafish embryos were dechorionated and anesthetized in 0.02% tricaine dissolved in Evans solution and dissected according to previously described procedures (Drapeau et al, 1999). Briefly, spinal neurons in somites 5-15 were selected for recording based on their soma size and position as visualized by oblique illumination (Olympus BX61W1).…”

“…Embryos were anesthetized in 0.02% Tricaine and immobilized in 0.75% low-melting point agarose. A total of 3 mM CNQX, 5 mM APV, 1 M TTX, or saline was coinjected with 0.1% Fast Green dye and 2% FITC cell-permeant fluorescent tracer dye, in Evans medium (134 mM NaCl, 2.9 mM KCl, 2.1 mM CaCl 2 , 1.2 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, pH 7.8, with NaOH) (Drapeau et al, 1999). Glass needles were pulled and mounted on a pipette holder, and 65 ms injections at 40 psi were delivered using a Picospritzer III (Parker Hannifin).…”

“…Electrophysiological recordings were done in the presence of 1-5 M strychnine to block glycinergic events, or in 10 M CNQX to block glutamatergic events. Patch-clamp electrodes for spinal neuron recordings (6 -14 M⍀) were pulled from borosilicate glass and were filled with the following intracellular solution (in mM): 105 D-gluconic acid, 16 KCl, 2 MgCl 2 , 10 HEPES, and 10 EGTA, adjusted to pH 7.2, 290 mOsm (Drapeau et al, 1999). Sulforhodamine B (0.1%; Sigma) was also included in the patch solution to label the cells and confirm their identity after a recording.…”