Publication Date

2010-05-08

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Physiology and Biophysics (Medicine)

Date of Defense

2010-04-16

First Committee Member

Wolfgang Nonner - Committee Chair

Second Committee Member

Grace Zhai - Committee Member

Third Committee Member

Ana Diez-Sampedro - Committee Member

Fourth Committee Member

Gerhard Dahl - Mentor

Fifth Committee Member

Daniela Boassa - Outside Committee Member

Abstract

Pannexins represent a recently discovered second family of gap junction proteins in vertebrates. However, instead of forming intercellular gap junction channels like connexins, pannexins operate as unpaired pannexons, allowing the flux of molecules from the cytoplasm to the extracellular space and vice versa. Pannexins appear to play a vital role in the local control loop of blood perfusion and oxygen delivery. The properties of Panx1 channels indicate that this protein is the most probable candidate for an ATP release channel and is involved in the propagation of intercellular calcium waves. It is also proposed to mediate the large pore formation of the P2X7 receptor death complex. Prolonged activation of this receptor can lead to cell death. There must be some mechanisms to stop this ATP-induced ATP release and opening of the lethal pore. Here we describe a negative feedback loop controlling pannexin 1 channel activity. ATP, permeant to pannexin 1 channels, was found to inhibit its permeation pathway when applied extracellularly. ATP analogues, including BzATP, suramine, and BBG were even more effective inhibitors of pannexin 1 currents than ATP. These compounds also attenuated the uptake of dyes by erythrocytes, which express pannexin 1. The rank order of the compounds in attenuation of pannexin 1 currents was similar to their binding affinities to the P2X7 receptor, except that receptor agonists and antagonists both were inhibitory to the channel. The ATP inhibitory effect is largely decreased when R75 on the first extracellular loop of Pannexin1 is mutated to alanine, strongly indicating that the ATP regulates this channel through binding. To further investigate the structural property of the ATP binding, we did alanine scanning mutagenesis of the extracellular loops and found that mutations on W74, S237, S240, I247 and L266 on the extracellular loops severely impair the BzATP inhibitory effect indicating that they might be direct binding partners for the ligands. Mutations on R75, S82, S93, L94, D241, S249, P259 and I267 have largely decreased BzATP sensitivity. Mutations on other residues didn't change the BzATP sensitivity compared to the wild type except for some nonfunctional mutants. All these data demonstrate that some amino acid residues on the extracellular loop of Pannexin 1 mediate ATP sensitivity. However, how these residues form the ATP-binding pocket remains to be elucidated.

Keywords

Pannexin 1; ATP; Negative Feedback; P2X7 Receptor

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