Functional reconstitution of bacterially expressed human potassium channels in proteoliposomes: membrane potential measurements with JC-1 to assay ion channel activity.
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.
Chanda
B
B
National Centre for Biological Sciences, TIFR Centre, Indian Institute of Science Campus, P.O. 1234, Bangalore 12, India.
Mathew
M K
MK
D015761:4-Aminopyridine / Q000494:pharmacology; D001419:Bacteria / Q000378:metabolism; D001562:Benzimidazoles; D002232:Carbocyanines; D003001:Cloning, Molecular; D005456:Fluorescent Dyes; D006801:Humans; D008081:Liposomes; D008564:Membrane Potentials; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry*; D011510:Proteolipids
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D008081:Liposomes; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011510:Proteolipids; C020485:proteoliposomes; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D015761:4-Aminopyridine