Abstract
The activation of large conductance, calcium-sensitive K(+) (BK(Ca)) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK(Ca) channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK(Ca) channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BK(Ca) channels and a catalytically inactive ("dead") mutant of human cGKIalpha prevented enhancement of BK(Ca) channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIalpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIalpha were found to associate with BK(Ca) channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIalpha was able to directly phosphorylate immunoprecipitated BK(Ca) channels, suggesting that cGKIalpha-dependent phosphorylation of BK(Ca) channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIalpha alone is sufficient to promote the enhancement of BK(Ca) channels in situ after activation of the NO/cGMP signaling pathway.
Highlights
The activation of large conductance, calcium-sensitive K؉ (BKCa) channels by the nitric oxide (NO)/cyclic GMP signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle
Earlier reports have shown that native HEK 293 cells contain an endogenous cyclic GMP (cGMP)-dependent protein kinase activity (15, 34, 35); our new data, described below, strongly suggest that this activity can be accounted for by the presence of the ␣ isoform of type I cGMPdependent protein kinase in these cells
We have examined the importance of the cGMP-dependent protein kinase (cGKI) in the regulation of a BKCa channel by the NO/cGMP signaling pathway in intact cells
Summary
The activation of large conductance, calcium-sensitive K؉ (BKCa) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. The exact mechanism(s) by which elevated cGMP causes smooth muscle relaxation has not been clearly defined, cGMP is known to influence a number of cellular processes (4), such as the levels of cytosolic free calcium, myosin light chain dephosphorylation (5), and the activity of voltagedependent, L-type calcium channels (6). In both vascular and nonvascular smooth muscle, activation of large conductance, calcium-sensitive Kϩ channels (maxi-K or BKCa channels) is reported to occur in response to endogenous NO or exogenous NO donors (7–12). BKCa channels appear to be important cellular effectors for the vasodilatory actions of the NO/cGMP signaling pathway because blockade of BKCa channels can interfere with the relaxationpromoting effects of NO (16 –18)
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