We propose to map the important structural binding domains of the two K+ transporters of heart mitochondria: the K+/H+ exchanger and the ATP- dependent K+ channel. Regulated K+ transport via these two porters is responsible for mitochondrial volume homeostasis and maintenance of the oxidative phosphorylation capacity of mitochondria. Both porters contain DCCD-reactive carboxyl residues that are involved in the translocation mechanism, as indicated by inhibition of transport by DCCD. We will use [14C]-DCCD and fluorescent DCCD analogs to label the sites on each porter and cyanogenbromide cleavage to obtain smaller peptides for sequencing. A similar approach will be used to label the ATP-dependent K+ channel with 2- azido-[32P]-ATP in the presence of Mg2+ to access the binding site for Mg- ATP on this protein. Moreover, the Mg2+-regulatory binding site on the K+/H+ exchanger and the Mg-ATP binding site on the ATP-dependent K+ channel will be studied using time resolved fluorescence of Mg2+ analogs such as Eu3+ and Tb3+. We will also use fluorescent analogs of DCCD to study conformational changes involved in transport regulation during Mg2+ binding.
