Tachykinin peptide-induced activation and desensitization of neurokinin 1 receptors☆
Introduction
The tachykinins are a group of neuropeptides characterized by the conserved C-terminal sequence, Phe–Xaa–Gly–Leu–Met–NH2 (Fig. 1). Substance P (SP) is the most well studied tachykinin and has been implicated in a number of physiological roles including inflammation, nociception, and regulation of smooth muscle. In addition to SP, several tachykinin peptides have been found to be active in invertebrates and in nonmammalian vertebrates, particularly amphibians [10], [11]. Three such peptides, ranatachykinin A (RTKA), ranatachykinin B (RTKB) and ranatachykinin C (RTKC), have been isolated from bullfrog (Rana catesbeiana) brain and gut [7].
To date the actions of the ranatachykinins (RTKs) on neuronal cells have not been studied. The RTKs actions were characterized using the classical pharmacology preparations of rat duodenum and guinea pig ileum [7]. Another study of several tachykinins, including RTKA, looked at peptide action on ion transport in the frog skin mediated by a neurokinin-1-like receptor [9]. We have previously reported the effects of the RTKs to produce Ca2+ elevations and receptor desensitization at the bullfrog substance P receptor (bfSPR) in a heterologous expression system [13]. In the present study, we have assessed the effects of the RTKs to modulate the M-current of sympathetic neurons from the bullfrog. This provides the first description of actions of the RTK peptides on native neurons.
The cDNA for the bfSPR is an orthologue of the mammalian neurokinin 1 receptor (NK1R) based on structural identity and ligand binding characteristics [16]. The rat (r)NK1R and bfSPR are 69% identical at the amino acid level, with the largest degrees of conservation seen in the transmembrane and intracellular loop regions. Nine of the ten proposed binding interactions between SP and the rNK1R are completely conserved in the bfSPR and the remaining binding site is likely conserved due to tertiary structural conditions caused by a disulfide bond in the extracellular domain.
In addition to characterizing the actions of the RTKs on neurons, we have correlated peptide binding properties and receptor activation with the ability of the peptide to cause receptor desensitization. Our lab has previously shown that a tachykinin’s ability to produce signaling is not directly proportional to its ability to produce desensitization [13], but it is not known if these differences are due to differences in binding properties of the RTKs, if these effects are the same in neurons as in the heterologous expression system or if these properties are specific for the bfSPR. In the present study we have examined the binding properties, signal activation and receptor regulation properties of four tachykinin peptides, SP, RTKA, RTKB and RTKC, at the bfSPR and rNK1R.
Section snippets
Whole cell electrophysiology
Single neurons were dissociated from bullfrog sympathetic ganglia [17]. Briefly, ganglia were dissected, treated with enzymes and stored in growth medium at 4 °C for 1–3 days. The ganglia were triturated to release single neurons for daily use. Under these conditions synaptic contacts do not form between isolated neurons.
Whole cell recordings of isolated neurons were conducted at room temperature, ∼23 °C, with a patch clamp amplifier (Warner PC-501, Hamden, CT). Peptides were dissolved in
Peptide inhibition of M-type potassium current
The effects of RTKA, RTKB and RTKC on native neurons have been assessed by measuring the change in IM following application of the peptides at various concentrations. Each of the RTKs resulted in an inhibition of IM. The concentration–response curves for inhibition of IM are presented in Fig. 3. Like SP, the RTKs inhibit IM in a concentration-dependent manner (, ) with similar maximum effects. The rank order of potency for inhibiting IM is with EC50 values
Discussion
Two major findings are presented here. First, the RTKs affect single bullfrog neurons by inhibiting the M-current. This is the first evidence that the RTKs regulate neuronal excitability by activating an endogenous tachykinin receptor. Second, we have studied the correlation between receptor binding, signal activation and the receptor regulation of desensitization and internalization. There is no direct correlation of a peptide’s ability to bind to the NK1R and activate second messenger
Conclusion
We have shown that the RTKs act as full agonists at an endogenous tachykinin receptor expressed in sympathetic ganglia neurons from the bullfrog. This interaction results in inhibition of a potassium current known as the M-current and likely causes neuronal excitability to be altered and fine-tuned. This response undergoes rapid desensitization thereby giving another mean of regulating cellular activity. These findings show that the RTKs, which were isolated from bullfrog brain and gut, act at
Acknowledgements
We would like to thank Dr. Gary Meszaros, Dr. James Krause and Ms. Anne Shriner for their assistance and contributions.
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The role of the N-terminal and mid-region residues of substance P in regulating functional selectivity at the tachykinin NK<inf>1</inf> receptor
2008, European Journal of PharmacologyDelineation of the motilin domain involved in desensitization and internalization of the motilin receptor by using full and partial antagonists
2007, Biochemical PharmacologyCitation Excerpt :From these data we can conclude that the ability of a peptide to induce desensitization is not solely determined by its ability to act as an agonist for signal activation events. Disparities between activation and desensitization have also been described for dopamine D1A receptors [29], μ-opioid receptors [30], the human tachykinin NK-1 [31] and serotonin 5-HT2C receptors [32]. Fusion of an EGFP protein to the carboxyl terminus of the MTLR did not seem to alter the pharmacological and functional properties of the MTLR.
Functional selectivity of NK<inf>1</inf> receptor signaling: Peptide agonists can preferentially produce receptor activation or desensitization
2006, Journal of Pharmacology and Experimental Therapeutics
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This work was supported by NIH grant NS25999 from the National Institute of Neurological Disorders and Stroke to MAS.