Voltage-dependent potassium channels are important for the proper functioning of excitable cells in the nervous system and in muscle. Different functional families of potassium channels are involved in modification and fine tuning of some of the excitable properties. Given the wide diversity of these channels and the crucial role they play in proper functioning of the excitable tissues, it is of great importance to understand the molecular basis of potassium channel diversity and function. Several members of one family, the A-type potassium channels, have been isolated as cDNA clones and expressed in Xenopus oocytes. Little is known about the biochemistry and especially the molecular biology of potassium channels from other families. Purification of potassium channels has been slow, and no sequence data is available for the design of synthetic oligonucleotides probes that could be used for routine cDNA isolation. Therefore, we have designed a sequence-dependent approach for the isolation of cDNAs encoding members of different potassium channel families. To achieve this, we use pools of different cDNA clones in a transcription- competent vector to synthesize mRNA in vitro. Expression of ion channels form transcripts in Xenopus oocytes and subdividing cDNA pools into """"""""cocktails"""""""" of smaller and smaller sizes has led to the isolation of a single cDNA clone encoding a new potassium channel. Using this strategy in combination with routine cloning procedures, we plan to isolate potassium channel clones that belong to different families. We plan to functionally characterize these isolates through expression in Xenopus oocytes. Comparison of amino acid sequences of members of different families will reveal conserved regions that may be important for general structure and function, diverged regions that may be involved in specific functions of some of the potassium channels. Such regions will be chosen as targets for site directed mutagenesis. Amino acid residues that may be functionally important will be altered. We shall introduce conservative changes in order not to perturb protein packing and conformation. Mutated cDNAs will be transcribed and in vitro prepared mRNA will be injected and expressed in Xenopus oocytes. Such a structure-function analysis will enable us to better understand the biophysics of ion channels and the molecular events underlying the phenomenon of excitability.
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