K+ CHANNELS IN THE BASOLATERAL MEMBRANE OF RAT CORTICAL COLLECTING DUCT ARE REGULATED BY A CGMP-DEPENDENT PROTEIN-KINASE

The basolateral membrane of the rat cortical collecting duct (CCD) principal cell is K+ conductive. Recently, two different K+ channels have been described, namely a small- and an intermediate-conductance K+ channel (s-K+ and i-K+) with most likely are responsible for the macroscopic K+ conductance. K+ channel activity was investigated at the single-channel level using the patch-clamp technique. Patch-clamp recordings were obtained from enzymatically isolated CCD segments and freshly isolated CCD cells using conventional cell-free, cell-attached, cell-attached-nystatin and slow-whole-cell methods. Both K+ channels showed rundown behaviour after excision. In an excised inside-out oriented membrane, K+ channels could be activated by simultaneous addition of 0.1 mmol/l MgATP to the bath. The i-K+ was activated in 13 out of 45, the s-K+ in 15 out of 45, cases. No activation of either channel was observed with cGMP alone (0.1 mmol/l), MgATP alone (0.1 mmol/l), cGMP and guanosine triphosphate (GTP) (0.1 mmol/l each) or cyclic adenosine monophosphate (cAMP) and MgATP (0.1 mmol/l each) n = 15, 11, 7, 8, respectively). The activated s-K+ could be blocked by KT 5823 (n = 8), a specific inhibitor of a cGMP-dependent protein kinase (PKG). An inhibition of the activated i-K+ was seen in seven cases. The membrane potential hyperpolarized significantly after application of dibutyryl-cGMP (0.1 mmol/l, n = 6) or nitroprusside (10 mu mol/l, n = 5), which is known to liberate NO and thus increase the intracellular cGMP level. In the presence of nitroprusside, the activity of the i-K+ on the cell was increased (n = 6). Furthermore, channel activity could be activated in the cell-attached configuration using calyculin A (10 nmol/l, n = 3), an indicate that both K+ channels are directly controlled by a membrane-bound PKG and a protein phosphatase.