MITOCHONDRIAL VDAC AND BACTERIAL PORA/C1: ION PERMEATION AND SELECTIVITY

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2005-07-25

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Abstract

VDAC and PorA/C1 are large diameter channels with properties reminiscent of those found in narrow channels. VDAC, located in the mitochondrial outer membrane, shows high selectivity for ATP over comparably sized ions. VDAC is characterized by a single open state with anionic selectivity and multiple cation-selective closed states. PorA/C1 from Neisseria meningitidis achieves high cationic selectivity and large conductance.

VDAC has multiple functions in cellular processes and the most important one is the regulation of metabolite flow across the outer membrane. A variety of functions could be achieved by the existence of different isoforms. In this thesis I summarized the electrophysiological properties of VDAC-like proteins from Drosophila Melanogaster encoded by genes CG17137, CG17139 and CG17140. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137, CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 did not form channels.

The special selectivity of VDAC for large anions was explored using the mutant of the mouse isoform 2. Inserted into planar membranes, mutant channels lack voltage gating, have a lower conductance, demonstrate cationic selectivity and, surprisingly, are still permeable to ATP. The estimated ATP flux through the mutant is comparable to that for the wildtype. Also we determined that the intact outer membrane containing the mutant is permeable to NADH and ADP/ATP. Both experiments support the counterintuitive conclusion that converting a channel from anion to cation preference does not substantially influence the flux of negatively charged metabolites. However, this finding supports the previous proposal that ATP translocation through VDAC is facilitated by a set of specific interactions between ATP and the channel wall.

The third part of my thesis represents experimental data supporting the theoretical model for the PorA/C1 structure. This model explains the almost ideal cationic selectivity of the channel and high level of rectification. These properties are proposed to arise from a high density of charges in the channel that results in both high selectivity and high ionic flux.

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