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 α-subunits, encoded by the slopoke gene (Slo), and one of four cell-specific accessory β-subunits (hβ1-4). We used patch-clamp analysis to determine the influence of hβ1, hβ2, and hβ4 on activation of human mesangial BK by cGMP kinase. We found that HEK 293 cells, coexpressing human (h) Sloα with either hβ1 or hβ2, contained single BK currents activated by db-cGMP in cell-attached patches. However, recombinant BK were not activated by db-cGMP when hSloα was expressed alone or with hβ4. DNA-RNA hybridization revealed that mesangial cells contained mRNA for hβ1 but not hβ2 or hβ4. The BK response to db-cGMP was decreased when hβ1 antisense but not scrambled oligonucleotides were incorporated into mesangial cells. Western blot analysis showed that hβ1 antisense oligonucleotide inhibited the amount of hβ1-V5 fusion protein expressed in HEK 293 cells by ∼50%. These results show that mesangial cells contain hβ1, a BK accessory protein, which confers activation of BK by cGMP kinase.
- Guanosine 3′,5′-cyclic monophosphate
- Human β-subunit
- Large-conductance calcium-activated potassium channels
- Patch clamp