Abstract
The P2X purinergic receptor channels (P2XRs) differ among themselves with respect to the rates of desensitization during prolonged agonist stimulation. Here we studied the desensitization of recombinant channels by monitoring the changes in intracellular free Ca(2+) concentration in cells stimulated with ATP, the native and common agonist for all P2XRs. The focus in our investigations was on the relevance of the P2XR C terminus in controlling receptor desensitization. When expressed in GT1 cells, the P2XRs desensitized with rates characteristic to each receptor subtype: P2X(1)R = P2X(3)R > P2X(2b)R > P2X(4)R > P2X(2a)R > P2X(7)R. A slow desensitizing pattern of P2X(2a)R was mimicked partially by P2X(3)R and fully by P2X(4)R when the six-amino acid sequences of these channels located in the cytoplasmic C terminus were substituted with the corresponding arginine 371 to proline 376 sequence of P2X(2a)R. Changing the total net charge in the six amino acids of P2X(4)R to a more positive direction also slowed the receptor desensitization. On the other hand, substitution of arginine 371-proline 376 sequence of P2X(2a)R with the corresponding sequences of P2X(1)R, P2X(3)R, and P2X(4)R increased the rate of receptor desensitization. Furthermore, heterologous polymerization of wild-type P2X(2a)R and mutant P2X(3)R having the C-terminal six amino acids of P2X(2a)R at its analogous position resulted in a functional channel whose desensitization was significantly delayed. These results suggest that composition of the C-terminal six-amino acid sequence and its electrostatic force influence the rate of receptor desensitization.
Highlights
Depending on the cell type examined [3]
Each subunit is proposed to have two putative transmembrane helices connected with a large extracellular loop, and both the N and C termini are located in the cytoplasm. From their N termini to the second transmembrane domain, the cloned subunits exhibit a relatively high level of amino acid sequence homology compared with their C termini, which are variable in lengths and show no apparent sequence homology except for the proximal region near the second transmembrane domain [1]
Based on the observed differences in their current desensitization kinetics, recombinant P2XRs are generally divided into two groups: the rapidly desensitizing (P2X1R and P2X3R) and the slowly desensitizing (P2X2R, P2X4R, P2X5R, P2X6R, and P2X7R) [3]
Summary
Vol 274, No 53, Issue of December 31, pp. 37651–37657, 1999 Printed in U.S.A. Contributions of the C-terminal Domain to the Control of P2X Receptor Desensitization*. Heterologous polymerization of wild-type P2X2aR and mutant P2X3R having the C-terminal six amino acids of P2X2aR at its analogous position resulted in a functional channel whose desensitization was significantly delayed. ATP-gated receptor channels (P2XRs) are expressed in a number of tissues where they are involved in regulation of a wide variety of cellular functions, including central and peripheral neurotransmission, smooth muscle contraction, platelet activation, and hormone secretion [1,2,3] The regulation of these functions by the activated P2XRs requires or is susceptible to local and/or global changes in intracellular Ca2ϩ concentration ([Ca2ϩ]i) [3]. We examined the importance of the C-terminal 6-aa sequences of P2XRs, which correspond to Arg371-Pro376 of P2X2aR (Fig. 1), to the desensitization pattern of these channels For this purpose, P2XRs and their mutants were expressed in GT1 immortalized neurons, and the impact of receptor desensitization on the pattern of [Ca2ϩ]i signaling was analyzed by monitoring single cell [Ca2ϩ]i.
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