Specific phosphorylation sites underlie the stimulation of a large conductance, Ca
            2+
            ‐activated K
            +
            channel by cGMP‐dependent protein kinase

The rate of urine secretion by insect Malpighian tubules (MTs) is regulated by multiple diuretic and antidiuretic hormones, often working either synergistically or antagonistically. In the Drosophila melanogaster MT, only diuretic factors have been reported. Two such agents are the biogenic amine tyramine (TA) and the peptide drosokinin (DK), both of which act on the stellate cells of the tubule to increase transepithelial chloride conductance. In the current study, TA and DK signaling was quantified by microelectrode recording of the transepithelial potential in isolated Drosophila MTs. Treatment of tubules with cGMP caused a significant reduction in the depolarizing responses to both TA and DK, while cAMP had no effect on these responses. To determine whether a specific cGMP-dependent protein kinase (PKG) was mediating this inhibition, PKG expression was knocked down by RNAi in either the principal cells or the stellate cells. Knockdown of Pkg21D in the stellate cells eliminated the modulation of TA and DK signaling. Knockdown of Pkg21D with a second RNAi construct also reduced the modulation of TA signaling. In contrast, knockdown of the expression of foraging or CG4839, which encodes a known and a putative PKG, respectively, had no effect. These data indicate that cGMP, acting through the Pkg21D gene product in the stellate cells, can inhibit signaling by the diuretic agents TA and DK. This represents a novel function for cGMP and PKG in the Drosophila MT and suggests the existence of an antidiuretic hormone in Drosophila .

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Stimulation of neuronal KATP channels by cGMP-dependent protein kinase: involvement of ROS and 5-hydroxydecanoate-sensitive factors in signal transduction

AJP Cell Physiology ◽

10.1152/ajpcell.00196.2009 ◽

2010 ◽

Vol 298 (4) ◽

pp. C875-C892 ◽

Cited By ~ 15

Author(s):

Yongping Chai ◽

Yu-Fung Lin

Keyword(s):

Protein Kinase ◽

Signaling Pathway ◽

Neuronal Excitability ◽

Single Channel ◽

Katp Channels ◽

Membrane Excitability ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein ◽

Stimulation Of

The ATP-sensitive potassium (KATP) channel couples intracellular metabolic state to membrane excitability. Recently, we demonstrated that neuronal KATP channels are functionally enhanced by activation of a nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling cascade. In this study, we further investigated the intracellular mechanism underlying PKG stimulation of neuronal KATP channels. By performing single-channel recordings in transfected HEK293 and neuroblastoma SH-SY5Y cells, we found that the increase of Kir6.2/SUR1 (i.e., the neuronal-type KATP) channel currents by PKG activation in cell-attached patches was diminished by 5-hydroxydecanoate (5-HD), an inhibitor of the putative mitochondrial KATP channel; N-(2-mercaptopropionyl)glycine, a reactive oxygen species (ROS) scavenger, and catalase, a hydrogen peroxide (H2O2)-decomposing enzyme. These reagents also ablated NO-induced KATP channel stimulation and prevented the shifts in the single-channel open- and closed-time distributions resulting from PKG activation and NO induction. Bath application of H2O2 reproduced PKG stimulation of Kir6.2/SUR1 but did not activate tetrameric Kir6.2LRKR368/369/370/371AAAA channels. Moreover, neither the PKG activator nor exogenous H2O2 was able to enhance the function of KATP channels in the presence of Ca2+ chelators and calmodulin antagonists, whereas the stimulatory effect of H2O2 was unaffected by 5-HD. Altogether, in this report we provide novel evidence that activation of PKG stimulates neuronal KATP channels by modulating intrinsic channel gating via a 5-HD-sensitive factor(s)/ROS/Ca2+/calmodulin signaling pathway that requires the presence of the SUR1 subunit. This signaling pathway may contribute to neuroprotection against ischemic injury and regulation of neuronal excitability and neurotransmitter release by modulating the function of neuronal KATP channels.

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Expression of Constitutively Active cGMP-Dependent Protein Kinase Prevents Glucose Stimulation of Thrombospondin 1 Expression and TGF- Activity

Diabetes ◽

10.2337/diabetes.52.8.2144 ◽

2003 ◽

Vol 52 (8) ◽

pp. 2144-2150 ◽

Cited By ~ 44

Author(s):

S. Wang ◽

X. Wu ◽

T. M. Lincoln ◽

J. E. Murphy-Ullrich

Keyword(s):

Protein Kinase ◽

Glucose Stimulation ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Thrombospondin 1 ◽

Dependent Protein ◽

Stimulation Of ◽

Constitutively Active

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Enhancement of the large conductance, Ca 2+ ‐activated K + (BK) channel current by cGMP‐dependent protein kinase requires multiple phosphorylation sites on the C‐terminal tail

The FASEB Journal ◽

10.1096/fasebj.26.1_supplement.870.18 ◽

2012 ◽

Vol 26 (S1) ◽

Author(s):

Barry D Kyle ◽

Soleil Hurst ◽

Andrew P Braun

Keyword(s):

Protein Kinase ◽

Bk Channel ◽

Phosphorylation Sites ◽

Dependent Protein Kinase ◽

Channel Current ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

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Stimulation of plasma membrane Ca2+-pump ATPase of vascular smooth muscle by cGMP- dependent protein kinase: Functional reconstitution with purified proteins

Muscle Physiology and Biochemistry ◽

10.1007/978-1-4615-5543-8_20 ◽

1999 ◽

pp. 157-167

Author(s):

Yutaka. Yoshida ◽

Akira Toyosato ◽

Mohammed Omedul Islam ◽

Takaki Koga ◽

Satoru Fujita ◽

...

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Protein Kinase ◽

Vascular Smooth Muscle ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein ◽

Stimulation Of

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The cGMP-dependent protein kinase stimulates the basolateral 18-pS K channel of the rat CCD

AJP Cell Physiology ◽

10.1152/ajpcell.2000.278.6.c1212 ◽

2000 ◽

Vol 278 (6) ◽

pp. C1212-C1217 ◽

Cited By ~ 23

Author(s):

Wen Hui Wang

Keyword(s):

Protein Kinase ◽

Collecting Duct ◽

Basolateral Membrane ◽

K Channel ◽

Channel Activity ◽

Patch Clamp Technique ◽

Dependent Protein Kinase ◽

Cyclic Monophosphate ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

We used the patch-clamp technique to study the effect of cGMP on the 18-pS K channel in the basolateral membrane of the rat cortical collecting duct. Addition of 100 μM 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) increased the activity of the 18-pS K channel, defined by NP o, by 95%. In contrast, applying 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) has no effect on channel activity. The effect of 8-Br-cGMP was observed only in cell-attached but not in inside-out patches. Application of 1 μM KT-5823, an inhibitor of the cGMP-dependent protein kinase (PKG), not only reduced the channel activity, but also completely abolished the stimulatory effect of 8-Br-cGMP, suggesting that the 18-pS K channel is not a cGMP-gated K channel. Addition of H-89, an agent that also blocks the PKG, mimicked the effect of KT-5823. To examine the possibility that the effect of 8-Br-cGMP is the result of inhibiting cGMP-dependent phosphodiesterase (PDE) and, accordingly, increasing cAMP or cGMP levels, we explored the effect on the 18-pS K channel of IBMX, an agent that inhibits the PDE. The addition of 100 μM IBMX had no significant effect on channel activity in cell-attached patches. Moreover, in the presence of IBMX, 8-Br-cGMP increased the channel activity to the same extent as that observed in the absence of IBMX, suggesting that the effect of cGMP is not mediated by inhibiting the cGMP-dependent PDE. That the effect of cGMP is mediated by stimulating PKG was further indicated by experiments in which application of exogenous PKG restored the channel activity when it decreased after the excision of the patches. In contrast, adding exogenous cAMP-dependent protein kinase catalytic subunit failed to reactivate the run-down channels. We conclude that cGMP stimulates the 18-pS channel, and the effect of cGMP is mediated by PKG.

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