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Alanine substitution scanning of pannexin1 reveals amino acid residues mediating ATP sensitivity

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Abstract

Pannexin1 is a prime candidate to represent an ATP release channel. The pannexin1 channel can be activated by extracellular ATP through purinergic receptors P2X7 or P2Y. Recent studies have shown that the Pannexin1 channel is inhibited by its own permeant ion, ATP, and also by P2X7 receptor agonists and antagonists. However, the dose dependence of this inhibition indicated that significant inhibition was prominent at ATP concentrations higher than required for activation of purinergic receptors, including P2X7 and P2Y2. The inhibitory effect of ATP is largely decreased when R75 in the first extracellular loop of Pannexin1 is mutated to alanine, indicating that ATP regulates this channel presumably through binding. To further investigate the structural property of the putative ATP binding site, we performed alanine-scanning mutagenesis of the extracellular loops of pannexin1. Mutations on W74, S237, S240, I247 and L266 in the extracellular loops 1 and 2 severely impaired the inhibitory effect of BzATP, indicating that they might be the essential amino acids in the putative binding site. Mutations on R75, S82, S93, L94, D241, S249, P259 and I267 moderately (≥50%) decreased BzATP sensitivity, suggesting their supporting roles in the binding. Mutations of other residues did not change the BzATP potency compared to wild-type except for some nonfunctional mutants. These data demonstrate that several amino acid residues on the extracellular loops of Pannexin1 mediate ATP sensitivity. Cells expressing mutant Panx1W74A exhibited an enhanced release of ATP, consistent with the removal of a negative feedback loop controlling ATP release.

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Abbreviations

Panx1:

Pannexin1

BzATP:

2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate

BBG:

Brilliant Blue G

MTSET:

[2-(trimethylammonium)ethyl] methanethiosulfonate bromide

MTSEA:

2-aminoethyl methanethiosulfonate hydrobromide

MTSES:

Sodium 2-sulfonatoethyl methanethiosulfonate

MBB:

Maleimidobutyryl-biocytin

CBX:

Carbenoxolone

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Acknowledgments

We thank Dr. Peter Larsson for reading an early version of the manuscript. This work was supported by NIH grant GM 48610.

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Authors and Affiliations

  1. Department of Physiology and Biophysics, University of Miami, School of Medicine, PO Box 016430, Miami, FL, 33101, USA

    Feng Qiu, Junjie Wang & Gerhard Dahl

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  1. Feng Qiu
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Correspondence to Gerhard Dahl.

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Qiu, F., Wang, J. & Dahl, G. Alanine substitution scanning of pannexin1 reveals amino acid residues mediating ATP sensitivity. Purinergic Signalling 8, 81–90 (2012). https://doi.org/10.1007/s11302-011-9263-6

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