Effect of extracellular potassium on the activity of distal renal tubule in mice / 上海交通大学学报（医学版）

Objective: To investigate the effect of extracellular potassium (K+) concentration on the activities of sodium chloride co-transporter (NCC) and large conductance Ca2+-activated K+ channel (BK) in distal renal tubule of mice. Methods: Six specific pathogen free (SPF) C57BL/6 mice aged 8 to 10 weeks were sacrificed,and the kidney slices were made with previously reported method. Then,these slices were incubated randomly in normal K+,high K+,BaCl2 and RbCl solutions,respectively. The abundance and phosphorylation level of NCC in kidney slices at different K+ concentrations and different time courses were detected by Western blotting. The overall and membrane expressions of BK in kidney slices were also detected after incubation with different K+ solutions for 2 h. Results: Compared with normal K+ solution,NCC phosphorylation level was significantly decreased after incubation with high K+ solution for 5,15,30 min (all P<0.05),and NCC phosphorylation level was also decreased after intervention with K+ channel inhibitor Ba2+ or Rb+ (both P<0.05). After the treatment with high K+ solution for 2 h,neither the overall cell expression of BKα subunit and β4 subunit,nor membrane expression of BKα subunit was found significant changes compared with normal K+ incubation. Conclusion: High K+ can directly down-regulate NCC phosphorylation level,which may be preparation for kaliuresis of the downstream tubule of distal convoluted tubule.

Construction of stable cell lines expressing large conductance Ca2+-activated K+ channel α-subunit and molecular mechanism of potassium excretion in this channel / 上海交通大学学报（医学版）

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.

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca2+ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca2+ concentration ([Ca2+]i), and the increase of [Ca2+]i by OxLDL or by LPC was inhibited by La3+ or heparin. LPC failed to increase [Ca2+]i in the presence of an antioxidant tempol. In addition, store-operated Ca2+ entry (SOC), which was evoked by intracellular Ca2+ store depletion in Ca2+-free solution using the sarcoplasmic reticulum Ca2+ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca2+]i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La3+ or U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca2+ to 1 micrometer activated large-conductance Ca2+-activated K+ (BKCa) current spontaneously, and this activated BKCa current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca2+-permeable Ca2+-activated NSC current and BKCa current simultaneously, thereby increasing SOC.

VOCC and BKCa mRNA expression in kidney tissues of IgA nephropathy patients / 上海第二医科大学学报

0.05). ConclusionThe expression of VOCC mRNA and BKCa mRNA in kidney tissues of IgA nephropathy patients are abnormal.There is positive correlation between the abnormal expression of VOCC mRNA and BKCa mRNA and total glomerular pathological lesions integrals.The expression of VOCC mRNA and BKCa mRNA in kidney tissues of IgA nephropathy may serve as the indictor for the disease progression.

Increased activity of large conductance Ca2+-activated K+ channels in negatively-charged lipid membranes

The effects of membrane surface charge originated from lipid head groups on ion channels were tested by analyzing the activity of single large conductance Ca2+-activated K+ (maxi K) channel from rat skeletal muscle. The conductances and open-state probability (Po) of single maxi K channels were compared in three types of planar lipid bilayers formed from a neutral phosphatidyledianolamine (PE) or two negatively-charged phospholipids, phosphatidylserine (PS) and phosphatidylinositol (PI). Under symmetrical KCl concentrations (3 apprx 1,000 mM), single channel conductances of maxi K channels in charged membranes were 1.1 apprx 1.7 times larger than those in PE membranes, and the differences were more pronounced at the lower ionic strength. The average slope conductances at 100 mM KCl were 251 +/- 9.9, 360 +/- 8.7 and 356 +/- 12.4 (mean +/- SEM) pS in PE, PS and PI membranes respectively. The potentials at which Po was 1/2, appeared to have shifted left by 40 mV along voltage axis in the membranes formed with PS or PI. Such shift was consistently seen at pCa 5, 4.5, 4 and 3.5. Estimation of the effect of surface charge from these data indicated that maxi K channels sensed the surface potentials at a distance of 8 apprx 9 ANG from the membrane surface. In addition, similar insulation distance (7 apprx 9 ANG) of channel mouth from the bilayer surface charge was predicted by a 3-barrier-2-site model of energy profile for the permeation of K+ ions. In conclusion, despite the differences in structure and fluidity of phospholipids in bilayers, the activities of maxi K channels in two charged membranes composed of PS or PI were strikingly similar and larger than those in bilayers of PE. These results suggest that the enhancement of conductance and Po of maxi channels is mostly due to negative charges in the phospholipid head groups.