The long-term goal of these experiments is to understand the molecular mechanisms of potassium accumulation in the yeast Saccharomyces cerevisiae, and the means by which intracellular potassium is regulated homeostatically. The experiments are prompted by the recent discovery that the two main transporter proteins involved, products of the TRK! and TRK2 genes, are sequence-similar to a major class of bacterial potassium channels. This finding has led to development of atomic scale-models for the two proteins (S.R. Durell & H.R. Guy; Biophysical Journal 77: 789-807, 1999) based on the crystal structure of one potassium channel, KcsA from $treptomyces lividans. The experiments will use yeast genetics/molecular biology to make several series of site-directed mutations in the TRK1 and TRK2 genes, in order to test predictions of these """"""""quasi-channel"""""""" structural models, in regard to the organization of trans-membrane topology (folding), location of potassium affinity and selectivity, location of a postulated co-ion pathway through the proteins, and intra-molecular salt-bridge formation. Functional assays will make use of recently refined techniques for membrane patch-recording in yeast (Bertl et al.; Europ. J. Physiol. 436:999, 1998), along with several types of ion-flux assays on intact yeast cells and protoplasts. The transport systems involved are thought to be H+-K+ cotransporters (symporters), functionally resembling many proton- or sodium- coupled substrate transporters in the so-called 12-TM class of molecules, found in all the biological kindgoms. But their peculiar sequence/structure suggests that their detailed mechanism of """"""""active"""""""" transport may differ in fundamental ways from that of the more familiar proteins. Because these proteins are native to fungi, but thus far not to animal systems, they offer the possibility for design of fungal-specific antibiotics having few or no side effects, on human tissues for example. And because they appear to have a quasi-channel structure and some rather strange properties for ion-cotransporters (Bihler et al., FEBSLetters 447:115-120), they may shed light on the important phenomenon of alternative carder and channel function in certain neurotransmitter transport systems.
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