Store-operated Ca2؉ channels (SOC) are expressed in cultured human mesangial cells and activated by epidermal growth factor through a pathway involving protein kinase C (PKC). We used fura-2 fluorescence and patch clamp experiments to determine the role of PKC in mediating the activation of SOC after depletion of internal stores by thapsigargin.

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(MC). The present study was performed to determine the specific isoform(s) of conventional PKC involved in activating SOC in MC. Fura 2 fluorescence ratiometry showed that the thapsigargin-induced Ca 2ϩ entry (equivalent to SOC) was significantly inhibited by 1 M Gö -6976 (a specific PKC␣ and ␤I inhibitor) and PKC␣ antisense treatment (2.5 nM for 24-48 h). However, LY-379196 (PKC␤ inhibitor) and 2,2Ј,3,3Ј,4,4Ј-hexahydroxy-1,1Ј-biphenyl-6,6Ј-dimethanoldimethyl ether (HBDDE; PKC␣ and ␥ inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single-channel analysis in the cell-attached configuration revealed that Gö -6976 and PKC␣ antisense significantly depressed thapsigargin-induced activation of SOC. However, LY-379196 and HBDDE did not affect the SOC responses. In inside-out patches, application of purified PKC␣ or ␤I, but not ␤II or ␥, significantly rescued SOC from postexcision rundown. Western blot analysis revealed that thapsigargin evoked a decrease in cytosolic expression with a corresponding increase in membrane expression of PKC␣ and ␥. However, the translocation from cytosol to membranes was not detected for PKC␤I or ␤II. These results suggest that PKC␣ participates in the intracellular signaling pathway for activating SOC upon release of intracellular stores of Ca 2ϩ

In vascular smooth muscle and glomerular mesangial cells, relaxing agents such as nitric oxide and atrial natriuretic peptide activate large-conductance Ca2+-activated K+ channels (BK) via the cGMP kinase pathway. BK are composed of pore-forming alpha-subunits, encoded by the slopoke gene (Slo), and one of four cell-specific accessory beta-subunits (hbeta1-4). We used patch-clamp analysis to determine the influence of hbeta1, hbeta2, and hbeta4 on activation of human mesangial BK by cGMP kinase. We found that HEK 293 cells, coexpressing human (h) Sloalpha with either hbeta1 or hbeta2, contained single BK currents activated by db-cGMP in cell-attached patches. However, recombinant BK were not activated by db-cGMP when hSloalpha was expressed alone or with hbeta4. DNA-RNA hybridization revealed that mesangial cells contained mRNA for hbeta1 but not hbeta2 or hbeta4. The BK response to db-cGMP was decreased when hbeta1 antisense but not scrambled oligonucleotides were incorporated into mesangial cells. Western blot analysis showed that hbeta1 antisense oligonucleotide inhibited the amount of hbeta1-V5 fusion protein expressed in HEK 293 cells by approximately 50%. These results show that mesangial cells contain hbeta1, a BK accessory protein, which confers activation of BK by cGMP kinase.

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