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Biochim Biophys Acta. 1999 Jan 12;1416(1-2):92-100.

Functional reconstitution of bacterially expressed human potassium channels in proteoliposomes: membrane potential measurements with JC-1 to assay ion channel activity.

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1
National Centre for Biological Sciences, TIFR Centre, Indian Institute of Science Campus, P.O. 1234, Bangalore 12, India.

Abstract

Structure-function studies on ion channels have been greatly facilitated by the cloning of cDNAs from a variety of sources. However, obtaining detailed structural information on these proteins requires overexpression, purification and reconstitution in a functionally competent form. In this communication, we report on the functional reconstitution of a human potassium channel, Kv1.4, overexpressed in bacteria. We have assessed the activity of these channels using a spectroscopic assay with a potential-sensitive dye, JC-1. The presence of ion channels renders proteoliposomes selectively permeable to potassium ions as monitored by measurements of transmembrane electrical potential. We have optimised conditions wherein a 12% change in the fluorescence signal of the carbocyanine dye JC-1 per 10 mV change in membrane potential is obtained. Using this assay, we find that the reconstituted protein is potassium selective and its activity is blocked by 4-aminopyridine, a known potassium channel blocker.

PMID:
9889332
[Indexed for MEDLINE]
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