STRUCTURE OF ATP SENSITIVE POTASSIUM CHANNELS

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DESCRIPTION (Adapted from the investigator's application): ATP-sensitive K+ channels, KATP, can be reconstituted from SUR1, a member of the ATP-binding cassette superfamily and KIR6.2, a member of the inward rectifier K+ channel family. The reconstituted channel is a moderate inward rectifier whose conductance is appropriately modulated by ATP/ADP, by sulfonylurea channel blockers and potassium channel openers. Preliminary data suggest SUR1 and KIR6.2 assemble with a 1:1 stoichiometry into large complexes, presumably KATP channels. The overall objective of the application is to test the hypothesis that KATP channels have a tetrameric architecture, (SUR1/KIR6.2)4. The specific objectives are: 1) To establish that SUR1 and KIR6.2 associate to form a heteromultimeric complex. Association is being monitored using his-tagged SUR1 and KIR6.2 subunits, by utilizing specific lectins that bind SUR1, by cophotolabeling with 125I-azidoglibenclamide and by the appearance of a complex glycosylation pattern of SUR1. 2) To determine the stoichiometry of the complex. This is being done using sedimentation velocity measurements to estimate the molecular size(s) of the complexes and by engineering and expressing active channel forming fusion proteins with defined stoichiornetries, e.g., SUR1-KIR6.2 and SUR1-(KIR6.2)2. 3) To determine the stoichiometry of active KATP channels. This is being done using a mutant of KIR6.2, N 160D, that confers strong rectification on reconstituted KATP channels. Co-expression of wildtype KIR6.2 with the N 160D mutation gives heteromeric channels whose properties, along with SUR1-N160D fusion channels, will be used to determine if active channels are tetrameric. 4) To start to map the domains of interaction between SUR1 and KIR6.2. This is being done by engineering KIR6.2/KIR3.4 chimeras to determine which parts of KIR6.2 are needed for association and which for formation of active channels. KIR3.4 is similar to 6.2, but does not associate or form active channels with SUR1. This work will provide a model for ion channel regulation by members of the ATP-binding cassette superfamily.