Two intracellular channels, the mitochondrial ATP-dependent potassium channel (mitoK[ATP]) and the mitochondrial Ca2+-dependent potassium channel (mitoKca), figure prominently in the process of ischemic preconditioning. Here we seek to determine the molecular identities of mitoKATp and mitoKca. Our approach employs time-honored, assay-driven, protein purification principles, in conjunction with new sensitive proteomic tools that include 2-dimensional gel electrophoresis (2DGE) and mass spectrometry (MS). To overcome the scarcity of these channels within the cell, we will isolate up to 10 g of mitochondria from pig liver, several orders of magnitude more mitochondria than have been used in previous studies of this type. Purification will involve multiple rounds of chromatography, including affinity chromatography to harness the biospecificity of these K+-channels. In the case of mitoK[ATP], we will exploit its interaction with ATP, whereas for mitoKca, we will exploit its high-affinity interaction with the scorpion venom protein, iberiotoxin. The robustness of the pooling strategy will stem from the use of redundant assays. Firstly, functional reconstitution of K+-channel current in giant proteoliposomes will provide the ultimate assessment of potassium channel activity. Secondly, we will also employ assays that are specific to mitoK[ATP] and mitoKca. Our ability to identify proteins in K+-channel-enriched fractions will be determined by the sensitive proteomic tools of peptide-mass fingerprinting (PMF) and tandem mass-spectrometry (MS/MS). Alternatively, if yields are sufficient, protein sequence will be obtained by Edman degradation. Structural information, in the form of primary amino acid sequence or PMF-MS/MS data, will subsequently guide the cloning of these channels. The fundamental properties of the newly-identified channels, including pharmacological specificity, will be assessed using patch-clamp analysis to measure K+-selective current in giant proteoliposomes. Molecular architecture will be studied with respect to native molecular weight, subunit stoichiometry and intersubunit contacts, through protein crosslinking experiments. Finally, we will identify any post-translational modifications or K+-channel-interacting proteins.
NIH ExPorter
NIH ExPorter