M-type (KCNQ2/3) potassium channels are suppressed by activation of G q/11 -coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to the multivalent A-kinase-anchoring protein AKAP150. Peptides that block AKAP150 binding to the KCNQ2 channel complex antagonize the muscarinic inhibition of the currents. A mutant form of AKAP150, AKAP(ΔA), which is unable to bind protein kinase C (PKC), also attenuates the agonist-induced current suppression. Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for the inhibition. Phosphorylation of KCNQ2 channels was increased by muscarinic stimulation; this was prevented either by coexpression with AKAP(ΔA) or pretreatment with PKC inhibitors that compete with diacylglycerol. These inhibitors also reduced muscarinic inhibition of M-current. Our data indicate that AKAP150-bound PKC participates in receptor-induced inhibition of the M-current.The M-current is a low-threshold, slowly activating potassium current that exerts negative control over neuronal excitability. Activation of G q/11 -coupled receptors suppresses the Mcurrent, creating a slow excitatory postsynaptic potential, enhancing excitability and reducing spike-frequency adaptation 1,2 . The M-type K + channel is a promising therapeutic target, as the channel blocker linopirdine acts as a cognition enhancer 3,4 , and the channel activator retigabine functions as an anticonvulsant 5,6 . M-type channels are heteromeric complexes of certain KCNQ-family potassium channel subunits (KCNQ2-5) [7][8][9][10] . KCNQ2 and KCNQ3 were the first members of this family identified as M-channel forming subunits 7 . The KCNQ3 subunit is a core component that co-assembles with KCNQ2, KCNQ4 and KCNQ5 to form functional M-type channels 10 . © 2003 Nature Publishing GroupCorrespondence should be addressed to N.H. (hoshin@ohsu.edu). Note: Supplementary information is available on the Nature Neuroscience website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2014 March 04. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAlthough the subunits that form M-type K + channels have been identified, the molecular details of the signaling pathways that lead to suppression of the M-currents upon receptor stimulation have not yet been fully defined 2,11 . We know that inhibition results from activation of G proteins of the G q/11 family, with the α-subunit as the active moiety 12,13 , and that a 'diffusible' messenger is involved. That is, the receptor/G protein complex can be physically remote from the channel 14,15 . Thus, closure most likely results from some product of phospholipase C activity. M-type channels can be closed by raising intracellular calcium 16 , and there is evidence that this might be a 'second messenger' for bradykinin 17 and nucleotides 18 , but not...
We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [ Sympathetic nerve excitation stimulates -adrenergic receptors on cardiac myocytes by release of noradrenaline, leading to an increase in the contractility. This cardiostimulant effect is traditionally thought to be mediated by an increase in Ca 2ϩ permeation resulted from cyclic AMP-dependent phosphorylation of voltage-gated ion channels (1, 2). Opening of phosphorylated L-type Ca 2ϩ channels (3) and tetrodotoxin-sensitive Na ϩ channels (4) (11)(12)(13)(14). However, no information on the concentration of cADP-ribose after -adrenoreceptor stimulation has yet been reported.Membrane-bound and cytosolic ADP-ribosyl cyclases constitutively synthesize cADP-ribose from -NAD ϩ (15-21). Formation of cADP-ribose is increased or decreased by stimulation of muscarinic acetylcholine receptors in a subtype-specific manner, and this is mimicked by GTP and blocked by bacterial toxins in NG108-15 neuronal cells (22). ADP-ribosyl cyclase thus seems to be coupled directly with neurotransmitter or hormone receptors via different G proteins in the surface membrane of these cells (23). The same control of cADP-ribose formation could be carried out by ventricular adrenergic receptors. To address this question, we measured ADP-ribosyl cyclase activity in crude membrane fractions of rat ventricular myocytes in the presence or absence of an adrenergic agonist and GTP. EXPERIMENTAL PROCEDURES Materials--[2,8 adenine-3 H]NAD ϩ (30.5 Ci/mmol) and [adenylate-32 P]NAD ϩ (800 Ci/mmol) were purchased from NEN Life Science Products. Cyclic ADP-ribose was obtained from either Yamasa Shoyu (Choshi, Japan) or Sigma, and nicotinamide guanine dinucleotide ϩ (NGD ϩ ) was from Sigma. Azide-free cholera toxin (CTx) was purchased from Funakoshi (Tokyo, Japan). Silica Gel 60 F 254 plastic TLC sheets were obtained from Merck.Membrane Preparation-Wistar rats used were new born to 4 weeks old. Ventricular heart muscles from cold-anesthetized rats were washed once in ice-cold phosphate-buffered saline. Minced myocytes were suspended in 10 mM Tris-HCl solution, pH 7.3, with 5 mM MgCl 2 (5 ml for each ventricle) at 4°C for 30 min. The suspension was homogenized in a Teflon glass homogenizer. The resultant homogenate was centrifuged at 4°C for 5 min at 1000 ϫ g to remove unbroken cells and nuclei. Crude membrane fractions were prepared by centrifugation (twice) of homogenates at 105,000 ϫ g for 15 min. The supernatant was removed, and the final pellet was dispersed in 10 mM Tris-HCl solution, pH 6.6. In each experiment, membranes were freshly prepared and used immediately for enzymatic reactions. In some experiments, rats were intraperitoneally injected with CTx (100 ng/g of body weight) 16 h before sacrifice.In addition, membranes were prepared from Chinese hamster ovary (CHO) cells stably transfected. To est...
Cyclic ADP-ribose (cADP-ribose) is a putative second messenger or modulator. However, the role of cADP-ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear. Here, we show that glutamate stimulates ADP-ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGluRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6-deficient mice showed no increase in the ADP-ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate-stimulated cyclase activity. GTP-c-S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP-ribosyl cyclase is a feature common to individual cloned mGluRs, we expressed each mGluR subtype in NG108-15 neuroblastoma · glioma hybrid cells. The glutamate-induced stimulation of the cyclase occurs preferentially in NG108-15 cells over-expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate-induced activation or inhibition of the cyclase activity was eliminated after pre-treatment with cholera or pertussis toxin, respectively. Thus, the subtype-specific coupling of mGluRs to ADP-ribosyl cyclase via G proteins suggests that some glutamate-evoked neuronal functions are mediated by cADP-ribose.
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