Functional Characterization of a Second Porin Isoform in Drosophila melanogaster: DMPORIN2 FORMS VOLTAGE-INDEPENDENT CATION-SELECTIVE PORES

doi:10.1074/jbc.M310572200

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Functional Characterization of a Second Porin Isoform in Drosophila melanogaster

DMPORIN2 FORMS VOLTAGE-INDEPENDENT CATION-SELECTIVE PORES^*

Rita Aiello ${ddagger}$ , Angela Messina ${ddagger}$ , Bettina Schiffler $§$ , Roland Benz $§$ , Gianluca Tasco¶||, Rita Casadio¶**, and Vito De Pinto ${ddagger}$ ${ddagger}$ ${ddagger}$

From the Department of Chemical Sciences, University of Catania, 95125 Catania, Italy, Lehrstuhl für Biotechnologie, Universität Würzburg, 97074 Am Hubland, Germany, and the ^¶Department of Biology, Biocomputing Unit, University of Bologna, 40126 Bologna, Italy

Mitochondrial porins or voltage-dependent anion-selective channelsare channel-forming proteins mainly found in the mitochondrialouter membrane. Genome sequencing of the fruit fly Drosophilamelanogaster revealed the presence of three additional porin-likegenes. No functional information was available for the differentgene products. In this work we have studied the function ofthe gene product closest to the known Porin gene (CG17137 codingfor DmPorin2). Its coding sequence was expressed in Escherichiacoli. The recombinant DmPorin2 protein is able to form channelssimilar to those formed by DmPorin1 reconstituted in artificialmembranes. Furthermore, DmPorin2 is clearly voltage-independentand cation-selective, whereas its counterpart isoform 1 is voltage-dependentand anion-selective. Sequence comparison of the two porin isoformsindicates the exchange of four lysines in DmPorin1 for fourglutamic acids in DmPorin2. We have mutated two of them (Glu-66and Glu-163) to lysines to investigate their role in the functionalfeatures of the pore. The mutants E163K and E66K/E163K are endowedwith an almost full inversion of the ion selectivity. Both singlemutations partially restore the voltage dependence of the pore.We found that an additional effect with the double mutant E66K/E163Kwas the restoration of voltage dependence. Protein structurepredictions highlight a 16 ${beta}$ -strand pattern, typical for porins.In a three-dimensional model of DmPorin2, Glu-66 and Glu-163are close to the rim of the channel, on two opposite sides.DmPorin2 is expressed in all the fly tissues and in all thedevelopmental stages tested. Our main conclusions are as follows.1) The CG17137 gene may express a porin with a functional rolein D. melanogaster. 2) We have identified two amino acids ofmajor relevance for the voltage dependence of the porin pore.

Received for publication, September 24, 2003 , and in revised form, March 29, 2004.

^* This work was supported in part by University of Catania GrantCOFIN2003058409_004 (to V. D. P.) and Deutsche ForschungsgemeinschaftProject Be 865/10 (to R. B.). The costs of publication of thisarticle were defrayed in part by the payment of page charges.This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

^|| Recipient of a fellowship for the Ministry for Instruction,University, Research project "Hydrolases from Thermophiles:Structure, Function, and Homologous and Heterologous Expression"and supported by a project on Functional Genomics (ConsiglioNazionale delle Ricerche).

^** Recipient of grants "Development and Implementation of Algorithmsfor Predicting Protein Structure" (Ministry for University,Research in Science and Technology), Molecular Genetics (ConsiglioNazionale delle Ricerche), and Postgenomics PNR 2001-2003, FIRBArticle 8.

To whom correspondence should be addressed: Dept. of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italia. Tel.: 39-095-7384244; Fax: 39-095-337036; E-mail: vdpbiofa{at}unict.it.