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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Molecular identification and characterization of three isoforms of tachykinin NK₁-like receptors in the cane toad Bufo marinus

¹School of Medical Sciences and ³School of Biotechnology and Biomolecular Sciences,, University of New South Wales, Sydney 2052; and ²Department of Pharmacology, University of Sydney, Sydney 2006, Australia

Submitted 23 January 2004 ; accepted in final form 13 May 2004

The tachykinin peptide bufokinin, isolated from the cane toad intestine, is important in intestinal and cardiovascular regulation in the toad. In this study, three tachykinin NK₁-like receptor isoforms, bNK₁-A, bNK₁-B, and bNK₁-C, encoding proteins of 309, 390, and 371 amino acids, respectively, were cloned from the toad brain and intestine. These isoforms differ only at the intracellular COOH terminus. The bNK₁-A and bNK₁-B isoforms are similar to the truncated and full-length forms of the mammalian NK₁ receptor, whereas bNK₁-C is unique and does not correspond to any previously described receptor. RT-PCR studies demonstrated that three isoform transcripts are widely distributed in the toad with high expression in gut, spinal cord, brain, lung, and skeletal muscle. When expressed in COS-7 cells, bufokinin showed similar high affinity (IC₅₀ 0.6–0.8 nM) in competing for ¹²⁵I-labeled Bolton-Hunter bufokinin binding at all receptors, but the binding affinities of substance P (SP) and neurokinin A (NKA) were very different at each isoform. When expressed in Xenopus oocytes, the truncated isoform, bNK₁-A, was inactive, whereas bNK₁-B and bNK₁-C produced changes in chloride current when stimulated by tachykinins (minimum concentrations: bufokinin, 0.1 nM; SP, 1 nM; and NKA, 10 nM). A marked desensitization of the response was seen to subsequent applications of tachykinins, as experienced by the mammalian NK₁ receptor. In summary, our study describing three isoforms of NK₁-like receptor from the toad suggests that the alternative splicing of NK₁ receptor is a physiologically conserved mechanism and raises a fundamental question as to the physiological role of each isoform.

bufokinin; tachykinin receptors; sequencing and cloning; Xenopus oocyte expression; amphibian

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