Mercer, Eric Alexander John; (1996) Expression, characterisation and structural analysis of the putative slow voltage-dependent potassium channel minK. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Minimal potassium channel (minK) protein induces very slowly activating voltage-dependent K+ currents in oocytes. Mutagenesis studies indicated that minK protein is the integral part of the minK channel, however, recent studies identified an additional protein required for functional channel activity. MinK protein consists of 130 amino acids with one putative transmembrane region and extra- and intracellular amino and carboxyl domains, respectively. Investigations so far have predominantly been concerned with the electrophysiological, pharmacological and regulatory aspects of minK channel, however, the molecular mechanism of K+ conduction is only likely to emerge once the structure of the channel protein is resolved. To study the structure of minK, synthetic peptides and recombinant proteins corresponding to the functional domains of minK and complete minK protein were synthesised for structural analysis in both aqueous and phospholipid environments, using Fourier-transform infrared and Circular Dichroism spectroscopy. Recombinant proteins corresponding to the carboxyl terminal domain and complete minK protein were expressed in E. coli and baculovirus/insect cell expression systems. Spectroscopic analysis, in combination with previous results, indicated that the amino terminal domain adopts predominantly random structures with its amino teminus inserted in the membrane in an a-helical conformation. Contrary to previous findings, synthetic peptide corresponding to the transmembrane domain took up a predominantly a-helical structure in membrane environments. Recombinant carboxyl terminal domain (62 residues) and synthetic peptide corresponding to the terminal region of the domain (44 residues) were insoluble in aqueous buffer, but adopted structures with similar secondary structure content in micelle environments, consisting of predominantly helical and β-sheet structures, with an additional minor random element for the synthetic peptide. Purification of recombinant minK protein, expressed using both E. coli and baculovirus/insect cell expression systems, from endogenous host cell proteins proved unsuccessful. In addition to structural studies, immunohistochemical techniques were applied to both infected insect cells expressing recombinant minK protein, and small intestinal tissues. In infected insect cells, minK protein was located in the plasma membrane; however, attempts to record minK channel activity proved unsuccessful, suggesting that minK protein, in accordance with the literature, requires an additional protein for functional channel activity. Furthermore, in both infected and control cells, a previously unreported minK-like, but pharmacologically distinct, channel activity was observed. In jejunum and ileum, minK protein was shown to be confined to the apical membrane of epithelial cells, a finding consistent with the distribution of the protein in the morphologically and functionally related renal and submandibular epithelial cells.

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