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Abstract
P2X receptors are simple polypeptide channels that mediate fast purinergic depolarizations in both nerve and muscle. Although the depolarization results mainly from the influx of Na(+), these channels also conduct a significant Ca(2+) current that is large enough to evoke transmitter release from presynaptic neurons. We sought to determine the molecular basis of this Ca(2+) conductance by a mutational analysis of recombinant P2X(2) receptors. Wild type and 31 mutant P2X(2) receptors were expressed in HEK-293 cells and studied under voltage-clamp. We found that the relative Ca(2+) permeability measured from the reversal potentials of ATP-gated currents was unaffected by neutralizing fixed charge (Asp(315), Asp(349)) near the mouths of the channel pore. By contrast, mutations that changed the character or side chain volume of three polar residues (Thr(336), Thr(339), Ser(340)) within the pore led to significant changes in P(Ca)/P(Cs). The largest changes occurred when Thr(339) and Ser(340) were replaced with tyrosine; these mutations almost completely abolished Ca(2+) permeability, reduced P(Li)/P(Cs) by about one-half, and shifted the relative permeability sequence of Cs(+), Rb(+), K(+), and Na(+) to their relative mobility in water. Our results suggest that the permeability sequence of the P2X(2) receptor arises in part from interactions of permeating cations with the polar side chains of three amino acids located in a short stretch of the second transmembrane domain.
Highlights
P2X receptors are a ubiquitous family (P2X1–P2X7) of ligandgated ion channels activated by physiological concentrations of extracellular ATP [1]
The largest changes occurred when Thr339 and Ser340 were replaced with tyrosine; these mutations almost completely abolished Ca2؉ permeability, reduced PLi/PCs by about one-half, and shifted the relative permeability sequence of Cs؉, Rb؉, K؉, and Na؉ to their relative mobility in water
Our results suggest that the permeability sequence of the P2X2 receptor arises in part from interactions of permeating cations with the polar side chains of three amino acids located in a short stretch of the second transmembrane domain
Summary
Molecular Biology and Heterologous Expression of Recombinant Receptors—Site-directed mutagenesis of acidic and polar amino acids of the rat P2X2 receptor was performed using the overlap-primer extension method [17]. Ionic permeability ratios were determined from the difference in reversal potentials of ATP-gated currents generated in a range of extracellular solutions. Extracellular solutions used to measure monovalent cation permeability ratios contained 1 mM Ca2ϩ and 1 mM Mg2ϩ to prevent the progressive change in cation permeability that sometimes occurs when ATP-gated currents are studied in extracellular solutions lacking divalent cations2 [19, 20]. We used 30 M ATP to evoke ligandgated current when the extracellular solution contained 1 mM Ca2ϩ. PX/PCs was calculated as PX/PCs ϭ exp(⌬VrevF/RT), where PX/PCs is the relative permeability of Xϩ to Csϩ, ⌬Vrev is the difference in the reversal potentials measured in the two solutions, F is Faraday’s constant, R is the universal gas constant, and T is the absolute temperature. The data were pooled, analyzed by one-way analysis of variance, and evaluated using the Fisher’s protected least significant difference post hoc test of Statview 5.0 (SAS Institute, Cary, NC)
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