Dual Regulation of R-Type CaV2.3 Channels by M1 Muscarinic Receptors

Byung-Chang Suh,정진영,Hae-Jin Kweon 한국분자세포생물학회 2016 Molecules and cells Vol.39 No.4

Voltage-gated Ca2+ (CaV) channels are dynamically modu-lated by G protein-coupled receptors (GPCR). The M1 muscarinic receptor stimulation is known to enhance CaV2.3 channel gating through the activation of protein kinase C (PKC). Here, we found that M1 receptors also inhibit CaV2.3 currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated CaV2.3 currents by ~two-fold. After the PMA-induced potentiation, stimulation of M1 receptors decreased the CaV2.3 currents by 52  8%. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is responsible for the muscarinic suppression of CaV2.3 currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of PI(4,5)P2 to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed CaV2.3 currents, by 53  3%. Next, dephosphorylation of both PI(4)P and PI(4,5)P2 to PI by PJ translocation further decreased the current by up to 66  3%. The results sug-gest that CaV2.3 currents are modulated by the M1 receptor in a dual mode—that is, potentiation through the activation of PKC and suppression by the depletion of membrane PI(4,5)P2. Our results also suggest that there is rapid turnover between PI(4)P and PI(4,5)P2 in the plasma membrane.

Dual Regulation of R-Type Ca_V2.3 Channels by M₁ Muscarinic Receptors

Jeong, Jin-Young,Kweon, Hae-Jin,Suh, Byung-Chang Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.4

Voltage-gated $Ca^{2+}$ ($Ca_V$) channels are dynamically modulated by Gprotein-coupled receptors (GPCR). The $M_1$ muscarinic receptor stimulation is known to enhance $Ca_V2.3$ channel gating through the activation of protein kinase C (PKC). Here, we found that $M_1$ receptors also inhibit $Ca_V2.3$ currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated $Ca_V2.3$ currents by ~two-fold. After the PMA-induced potentiation, stimulation of $M_1$ receptors decreased the $Ca_V2.3$ currents by $52{\pm}8%$. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate ($PI(4,5)P_2$) is responsible for the muscarinic suppression of $Ca_V2.3$ currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of $PI(4,5)P_2$ to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed $Ca_V2.3$ currents, by $53{\pm}3%$. Next, dephosphorylation of both PI(4)P and $PI(4,5)P_2$ to PI by PJ translocation further decreased the current by up to $66{\pm}3%$. The results suggest that $Ca_V2.3$ currents are modulated by the $M_1$ receptor in a dual mode-that is, potentiation through the activation of PKC and suppression by the depletion of membrane $PI(4,5)P_2$. Our results also suggest that there is rapid turnover between PI(4)P and $PI(4,5)P_2$ in the plasma membrane.

M1 Muscarine성 수용체에서 123Arginine 잔기의 Site--mutagenesis가 신호전달계에 미치는 영향

이석용(Seok Yong Lee) 대한약학회 2000 약학회지 Vol.44 No.1

An exceptionally conserved sequence that is shared among most G protein-coupled neurotransmitter receptors is an aspartate-arginine-tyrosine triplet that is located at the second cytoplasmic domain. Using the m1 subtype of muscarinic acetylcholine receptors as an example, a point mutation of the arginine residue at position 123 into asparagine was induced. This mutation resulted in a complete blockade of the carbachol-induced increases of PI hydrolysis and intracellular Ca2+ level, in spite of the expression of the wild-type and mutant receptors at similar concentrations in Chinese hamster ovary cells. In marked contrast, the muscarinic agonist carbachol induced concentration-dependent enhancement of the activity of NO synthase at mutant m1 receptors although the enhancement was significantly smaller than at wild-type m1 receptors. These data suggest that this highly conserved arginine residue plays an important role in coupling of muscarinic receptors to the second messenger systems and the presence of alternate mechanisms of activation of neuronal NO synthase which might be operative in the absence of large changes in the concentration of cellular Ca2+.

Relaxing Effect of Acetylcholine on Phenylephrine-Induced Contraction of Isolated Rabbit Prostate Strips Is Mediated by Neuronal Nitric Oxide Synthase

Hoai Bac Nguyen,이신영,박수현,이무열,장인호,명순철 대한비뇨의학회 2013 Investigative and Clinical Urology Vol.54 No.5

Purpose: The location of acetylcholinesterase-containing nerve fibers suggests a role for acetylcholine in both contractility and secretion in the prostate gland. The colocalization of nitrergic nerves with cholinergic nerves, and the cotransmission of nitric oxide with acetylcholine in cholinergic nerves, has been demonstrated in the prostate glands of various species. Thus, we investigated the effects of acetylcholine on phenylephrine-induced contraction and the correlation between cholinergic transmission and nitric oxide synthase by using isolated prostate strips of rabbits. Materials and Methods: Isolated prostate strips were contracted with phenylephrine and then treated with cumulative concentrations of acetylcholine. Changes in acetylcholine-induced relaxation after preincubation with NG-nitroarginine methyl ester,7-nitroindazole, and aminoguanidine were measured. The effects of selective muscarinic receptor antagonists were also evaluated. Results: In the longitudinal phenylephrine-contracted strip, the cumulative application of acetylcholine (10-9 to 10-4 M) elicited a concentration-dependent relaxation effect. Acetylcholine-induced relaxation was inhibited not only by nitric oxide synthase inhibitors (10 μM L-NAME or 10 μM 7-nitroindazole) but also by 10 μM atropine and some selective muscarinic receptor antagonists (10-6 M 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one and 10-6M 4-diphenylacetoxy-N-methyl-piperidine). In contrast, relaxation was significantly increased by pretreatment of the strips with 10 mM L-arginine. Conclusions: Acetylcholine relaxed phenylephrine-induced contractions of isolated rabbit prostate strips. This relaxation may be mediated via both cholinergic and constitutive nitric oxide synthase with both the M2 and M3 receptors possibly playing key roles.

Changes in the Muscarinic Receptors on the Colonic Smooth Muscles of Rats with Spinal Cord Injury

주민철,김용성,최을식,오정택,Hyun Joon Park,이문영 대한재활의학회 2011 Annals of Rehabilitation Medicine Vol.35 No.5

Objective To investigate changes in (1) the colonic response to acetylcholine (Ach), (2) the muscarinic (M)receptors in the colon, and (3) the levels of colonic contraction-related proteins after a spinal cord injury (SCI). Method We divided 16 Sprague-Dawley rats into 2 groups: the control group and the SCI group. A spinal cord transection was performed surgically at the T10 vertebral level. After 1 week, the entire colon was divided into 2segments, the proximal and distal colon. Each segment was mounted in a longitudinal or circular muscle direction in a 10-ml organ bath. We determined the intergroup differences as percentage changes in contractility after Ach treatment alone, Ach treatment with M2 receptor antagonist (AQ-RA741) pretreatment, and Ach treatment with M3 receptor antagonist (4-DAMP) pretreatment. Western blot analyses were performed to determine the expression level of RhoA, and heat shock protein 27 (HSP27). Results Compared to the control rats, the SCI rats showed an increased response to Ach along both the directions in the proximal colon (p<0.05). Compared to the control group, in the SCI group, the Ach response was signifi cantly diff erent in the proximal segment under AQ-RA741 pretreatment (p<0.05) and in the distal segment under 4-DAMP pretreatment (p<0.05). Findings of the western blot analyses showed a signifi cant decrease in the level of protein gene product 9.5 in the proximal and distal colon and a signifi cant increase in the level of RhoA and HSP27 in the proximal colon of the SCI rats. Conclusion Our results suggest that changes in colonic contractility after SCI are partly attributable to changes in the M receptor subtypes.

천식 환자에서 M2 무스카린성 수용체 기능에 관한 연구

권영환 ( Kwon Yeong Hwan ),이상엽 ( Lee Sang Yeob ),박상면 ( Park Sang Myeon ),이신형 ( Lee Sin Hyeong ),신철 ( Sin Cheol ),조재연 ( Jo Jae Yeon ),심재정 ( Sim Jae Jeong ),강경호 ( Kang Gyeong Ho ),유세화 ( Yu Se Hwa ),인광호 ( I 대한결핵 및 호흡기학회 2000 Tuberculosis and Respiratory Diseases Vol.49 No.4