A Catalytically Inactive Mutant of Type I cGMP-dependent Protein Kinase Prevents Enhancement of Large Conductance, Calcium-sensitive K+ Channels by Sodium Nitroprusside and cGMP

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 ␣ isoform of type I cGMP-dependent protein kinase (cGKI). We observed that cotransfection of cells with BK Ca channels and a catalytically inactive ("dead") mutant of human cGKI␣ 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 cGKI␣ supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKI␣ were found to associate with BK Ca channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKI␣ was able to directly phosphorylate immunoprecipitated BK Ca channels, suggesting that cGKI␣-dependent phosphorylation of BK Ca channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKI␣ alone is sufficient to promote the enhancement of BK Ca channels in situ after activation of the NO/cGMP signaling pathway.The elevation of intracellular cGMP 1 in response to endothelium-derived nitric oxide (NO) or clinically prescribed nitrovasodilators, such as nitroglycerin and sodium nitroprusside, is known to play an important role in the hypotensive actions of these agents (1, 2). Similarly, elevation of cGMP by the phosphodiesterase inhibitor sildenafil (Viagra) (3) appears to underlie the smooth muscle-relaxing and anti-impotence effects of this drug. Although 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 K ϩ channels (maxi-K or BK Ca channels) is reported to occur in response to endogenous NO or exogenous NO donors (7-12). In many cases, addition of exogenous cGMP appears to mimic the effects of NO and NO donors on BK Ca channel activation (8, 10, 12-15), suggesting that cGMP acts downstream of NO. Physiologically, BK Ca channels appear to be important cellular effectors for the vasodilatory actions of the NO/cGMP signaling pathway because blockade of BK Ca channels can interfere with the relaxationpromoting effects of [16][17][18].A major intracellular target for cGMP in smooth muscle...

Hypercontractility and impaired sildenafil relaxations in the BKCachannel deletion model of erectile dysfunction

“…Thus sildenafil acts on the NO/sGC/ cGMP pathway and amplifies its relaxing effect in smooth muscle. cGMP-dependent protein kinase directly activates the BK Ca channel (32,36), and indirectly through phosphorylation of phospholamban and elevation of local Ca 2ϩ release events through ryanodine receptors (30). We found that the relaxing effect of sildenafil (10 M) was reduced by ϳ10% in Slo Ϫ/Ϫ CCSM.…”

DAF-FM (4-Amino-5-methylamino-2′,7′-difluorofluorescein) Diacetate Detects Impairment of Agonist-Stimulated Nitric Oxide Synthesis by Elevated Glucose in Human Vascular Endothelial Cells: Reversal by Vitamin C and l-Sepiapterin

“…Protein samples were incubated for ϳ5 min at 95°C and then resolved by denaturing SDS-PAGE using a 9% separating gel. After overnight transfer of resolved proteins to nitrocellulose membrane (18 V, 4°C), Western blotting was performed using an anti-human eNOS monoclonal antibody (1:1000 dilution) (BD Biosciences, Mississauga, Ontario, Canada), as we have earlier described for other proteins (Swayze and Braun, 2001).…”

RACK1 is a BKCa channel binding protein

“…Given the BK Ca channel's role as an important regulator of Ca 2ϩ -dependent processes, it is perhaps not surprising that the BK Ca channel itself is regulated by second messengers in addition to calcium, including protein phosphatases (8,55,56), heterotrimeric GTP binding proteins (18,19), and protein kinases (36 -38). The BK Ca channel's pore-forming ␣-subunit is phosphorylated by cAMP-dependent protein kinase (PKA) (50,58), protein kinase C (PKC) (38), cGMP-dependent protein kinase (PKG) (3,48) and cSrc (24). The channel's auxiliary ␤ 1 -subunit (primarily expressed in smooth muscle) contains a clear PKG consensus sequence and a potential PKA site (51).…”

“…Thus, at least in certain tissues, electrophysiological experiments suggest that PKG and PKC stably associate with the BK Ca channel. Indeed, cSrc, PKG, and PKA have also been shown biochemically to associate with the BK Ca channel in either native tissues or heterologous expression systems (2,48,49,53,59), as has the Ca-dependent phosphatase calcineurin (25). Little is known, however, about how these proteins are organized into a regulatory complex.…”