Interaction of the neuropeptide substance P with nicotinic acetylcholine receptors of Torpedo electric organ

The noncompetitive inhibition of the activation of the nicotinic acetylcholine receptors (nAChRs) of Torpedo electroplaque by the neuropeptide substance P was examined. Carbamylcholine-stimulated {dollar}sp{lcub}22{rcub}{dollar}Na{dollar}sp+{dollar} efflux was used to assess the functional state of the receptor. Substance P inhibited the {dollar}sp{lcub}22{rcub}{dollar}Na{dollar}sp+{dollar} flux in a time- and concentration-dependent manner. Irreversible blockade of the nAChR by {dollar}alpha{dollar}-bungarotoxin ({dollar}alpha{dollar}Bgt) (1) shifted the dose-response curve for the substance P blockade of {dollar}sp{lcub}22{rcub}{dollar}Na{dollar}sp+{dollar} permeability to lower concentrations, and (2) decreased the steady state amount of {dollar}sp{lcub}22{rcub}{dollar}Na{dollar}sp+{dollar} released in proportion to the fraction of receptor activated without changing the time course of the ion flux, suggesting that substance P is capable of blocking the channel as well as increasing receptor desensitization.; Substance P increased the rate and extent of the carbamylcholine-induced increase in the agonist affinity, a phenomenon known as desensitization. But the kinetics of recovery after exposure to carbamylcholine plus substance P suggest that the effects of substance P may be dual: At low concentrations substance P enhances the receptor desensitization while at high concentrations it prevents the receptor from entering into the desensitized state. The existence of two different substance P binding sites having opposite effects on the receptor desensitization was further confirmed by the equilibrium binding of (3H) substance P: A high affinity site with a {dollar}Ksb{lcub}rm d{rcub}{dollar} of 0.5 {dollar}mu{dollar}M and a {dollar}Bsb{lcub}rm max{rcub}{dollar} of 1 per receptor and a second class of low affinity sites with a {dollar}Ksb{lcub}rm d{rcub}{dollar} of 36-72 {dollar}mu{dollar}M and a {dollar}Bsb{lcub}rm max{rcub}{dollar} of 7-14 per receptor. The high affinity site was exposed only in the presence of nicotinic agonist, consistent with its location within the low channel pore, while the low affinity sites were constitutively present.; The structure-activity relationships of the high affinity substance P site revealed a similar pharmacology to those of neuronal or muscle-type nAChRs, i.e., COOH-terminus of the peptide was important for the activity but nonmammalian tachykinins were totally inactive.; The high affinity binding site for substance P was covalently labeled using the photoreactive substance P derivative and a bifunctional crosslinker. The assignment of the location of the high affinity substance P binding site to the {dollar}alpha{dollar}-{dollar}delta{dollar} interface is consistent with the mechanism by which substance P enhances the affinity of ACh, i.e., the affinity of one of the two different ACh binding sites is selectively enhanced by substance P.; Overall, the substance P binding sites characterized here are associated with the inhibitory effects of substance P on the nAChR-mediated biological activities such as ion flux and catecholamine secretion. This result also suggests that the nAChR is prone to blockade by small cationic peptides as well as inorganic compounds of which biological significance is unknown.