ABSTRACT
Objective: To construct stable cell lines expressing the large conductance Ca2+-activated K+ channel (MaxiK or BK) α-subunit and to explore the mechanism of potassium excretion via BKα channel. Methods:The BKα plasmid with Myc tag was constructed and transfected into HEK293 cell lines by lipofectamine 2000. The positive monoclonal cell lines were screened by G418, and the expression of BKα was detected by Western blotting and the location of BKα by immunofluorescence. The stable cell lines expressing BKα protein was cultured on slides to form a single cell layer, which was perfused with different potassium ion concentrations of 5 mmol/L and 100 mmol/L, and the single channel patch clamp recorded the ion flux of BKα. Wild type and mutants (G77R, G130R, C140R and R297C) of the inwardly rectifying potassium channel (Kir4.1) were transfected into HEK293 cells stably transfected with BKα, and then the membrane protein was extracted. The expression of BKα was detected by Western blotting. Results:Stable cell lines expressing BKα channel were selected from HEK293 cells after transfection and cellular immunofluorescence verified the expression of BKα channel and its expression on the cell membrane. The channel open frequency (Npo) of BKα increased rapidly when perfused with 100 mmol/L potassium. After being transfected with wild type or mutants of Kir4.1, the membrane expression of BKα in the stable cell lines showed significant difference among these groups (P<0.05). Conclusion:The HEK293 cell lines stably expressing BKα have been successfully constructed. BKα channel can be activated by high potassium solutions. The function of the BKα subunit can be related to Kir4.1 channel, which may be attributed to the depolarization of the cells transfected by Kir4.1 mutants.
ABSTRACT
Effects of cadmium exposure on renal Na+ and K+ transports were studied in rats. During the course of cadmium treatment (2 mg Cd/kg/day, s.c. injections for 3 weeks) renal tubular transports of Na+ and K+ were evaluated by lithium clearance technique. During the early phase (first week) of cadmium treatment, urinary Na+ excretion decreased drastically and this was due to an increased Na+ reabsorption both in the proximal and distal nephrons. During the late phase (third week) of cadmium treatment, filtered Na+ load was decreased by reduction in GFR, but the renal Na+ excretion returned to the control level due to impaired Na+ transport in the proximal tubule. Urinary excretion of K+ did not change during the early phase, but it rose markedly during the late phase of cadmium treatment. These results indicate that a light cadmium intoxication induces a Na+ retention, and a heavy intoxication results in a K+ loss. Possible mechanisms for these changes are discussed.