Publication Date

2015-06-19

Availability

Open access

Embargo Period

2015-06-19

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Physiology and Biophysics (Medicine)

Date of Defense

2015-06-15

First Committee Member

Gerhard Dahl

Second Committee Member

Laura Bianchi

Third Committee Member

Robert Keane

Fourth Committee Member

Kenneth Muller

Fifth Committee Member

Mary Lou King

Sixth Committee Member

Silvia Penuela

Abstract

Pannexin 1 is one of three pannexins, a vertebrate protein family related to the invertebrate innexins. Unlike its innexin relatives, Pannexin 1 does not form gap junctions in vivo, but it does form plasma membrane channels that act as ATP release channels. ATP release through Pannexin 1 serves multiple physiological roles throughout the body, including in the central nervous system, or CNS. Pannexin 1 also plays a pathophysiological role in CNS injury as part of the cell death-inducing P2X7-Pannexin 1 complex. During CNS injury, increased extracellular K+ activates Pannexin 1 by an unknown mechanism. Since Pannexin 1 block might ameliorate CNS injury, a selective Pannexin 1 blocker must be identified in order to target Pannexin 1 without causing side effects. Evidence from our lab indicates that when K+ activates Pannexin 1, it likely binds to an extracellular site on Pannexin 1. Potassium also activates an unknown mechanism that slowly increases Pannexin 1-mediated conductance in Xenopus oocytes. I systematically mutated extracellular residues of Pannexin 1 to alanine to find where K+o affects Pannexin 1. Alanine replacement of residues R75, S82, or L94 nearly abolished K+-mediated activation of Pannexin 1, and alanine replacement of residues W74, S249, P259, or I267 also diminished the stimulatory effect of K+ on Pannexin 1. Alanine replacements of S237, D241, or L266 boosted the stimulatory effect of K+ on Pannexin 1. All ten of these putative K+ binding site residues are also part of the putative ATP binding site; hence, the extracellular ATP and K+ binding sites overlap considerably. Overlapping binding sites support competition between ATP (the antagonist) and K+ (the agonist) on Pannexin 1. I tested the effects of solutions containing ATP (or the more potent analogue BZATP) and K+ on Pannexin 1 and found that increased concentrations of K+o abrogate the inhibitory effects of ATP or BZATP. This finding shows that K+ and ATP have competing and opposing effects on Pannexin 1. Override of ATP-dependent negative feedback by K+o is detrimental to CNS cells, since potassium-mediated Pannexin 1 activation promotes programmed cell death (including apoptosis). I searched for a more selective Pannexin 1 inhibitor and found that the food dyes Brilliant Blue FCF (BBFCF) and Fast Green FCF inhibit Pannexin 1 without inhibiting P2X7. I found other artificial and natural food dyes that also inhibit Pannexin 1, so dyes may be a new class of Pannexin 1 blockers to investigate. The binding sites of BBFCF and Fast Green FCF overlap with the ATP and K+ binding sites at residues W74, S237, and L266. This overlap might indicate competition between K+ and BBFCF/Fast Green FCF. 75 mM extracellular K+ overrides the inhibitory effects of BBFCF and Fast Green FCF. Fast Green FCF inhibits Pannexin 1 channels activated by 10 or 40 mM K+o. I concluded that BBFCF and Fast Green FCF compete with K+o. The results of our experiments and the experiments of our collaborators indicate that K+ binds to Pannexin 1, activates the channel, stimulates programmed cell death, and overrides the effects of ATP and Pannexin 1-inhibiting dyes.

Keywords

Pannexin 1; extracellular potassium; ATP; apoptosis; food dyes

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