ISSN:
1432-1424
Keywords:
Ca-activated K+ channel
;
solubilization
;
reconstitution
;
thick ascending limb of Henle's loop
;
calmodulin inhibitors
;
trypsin
;
pH
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Summary A barium-sensitive Ca-activated K+ channel in the luminal membrane of the tubule cells in thick ascending limb of Henle's loop is required for maintenance of the lumen positive transepithelial potential and may be important for regulation of NaCl reabsorption. In this paper we examine if the K+ channel can be solubilized and reconstituted into phospholipid vesicles with preservation of its native properties. The K+ channel in luminal plasma membrane vesicles can be quantitatively solubilized in CHAPS at a detergent/protein ratio of 3. For reconstitution, detergent is removed by passage over a column of Sephadex G 50 (coarse). K+-channel activity is assayed by measurement of86Rb+ uptake against a large opposing K+ gradient. The reconstituted K+ channel is activated by Ca2+ in the physiological range of concentration (K1/2∼2×10−7 m at pH 7.2) as found for the K+ channel in native plasma membrane vesicles and shows the same sensitivity to inhibitors (Ba2+, trifluoperazine, calmidazolium, quinidine) and to protons. Reconstitution of the K+ channel into phospholipid vesicles with full preservation of its native properties is an essential step towards isolation and purification of the K+-channel protein. Titration with Ca2+ shows that most of the active K+ channels in reconstituted vesicles have their cytoplasmic aspect facing outward in contrast to the orientation in plasma membrane vesicles, which requires also addition of Ca2+ ionophore in order to observe Ca2+ stimulation. The reconstituted K+ channel is highly sensitive to tryptic digestion. Brief digestion leads to activation of the K+ channel in absence of Ca2+, to the level of activity seen with saturating concentrations of Ca2+. This tryptic split is located in a cytoplasmic aspect of the K+ channel that appears to be involved in opening and closing the K+ channel in response to Ca2+ binding.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01869155
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