REGULATION OF SPONTANEOUS OPENING OF MUSCARINIC K+ CHANNELS IN RABBIT ATRIUM
REGULATION OF SPONTANEOUS OPENING OF MUSCARINIC K+ CHANNELS IN RABBIT ATRIUM
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DOI：
10.1113/jphysiol.1991.sp018445
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发表时间：
1991-02-01
影响因子：
5.5
通讯作者：
GILES, W
中科院分区：
文献类型：
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作者：
KAIBARA, M;NAKAJIMA, T;GILES, W
1.应用膜片钳技术分离单个肌细胞，研究了在没有M受体激动剂的情况下，兔心房肌细胞内控制M受体K+通道开放的机制(S)。在细胞贴附的膜片结构中，记录到了由M胆碱K+通道和内向整流K+通道活动引起的电流。然而，虽然只在被检查的211个斑块中观察到10个斑块的内向整流钾通道电流，但在这些心房细胞中所有被检查的斑块中都观察到了自发开放(即在没有毒鼠碱激动剂的情况下)的毒鼠碱K+通道电流。根据其电导和门控特性与吸管中含有0.5-mU-M-乙酰胆碱和膜片内侧存在10-mU-M-GTP时记录到的么正事件非常相似，确定了由M+通道自发开放引起的单通道电流。尽管在膜片切除后不久，毒扁豆碱K+通道的自发开放消失，但在将ATP和MgCl2应用于撕裂的斑片内侧后，这种类型的通道活动重新出现，正如先前的报道所预期的那样。ATP和镁离子(通道数N乘以开放概率P(O)的乘积)引起的K+通道活动强烈依赖于游离镁离子的浓度；然而，100mU-M-鸟苷5‘-O-3-硫代三磷酸(GTP-γ-S)结合三磷酸腺苷和镁离子激活M通道后，镁离子浓度从5.5×10~(-5)M增加到2×10~(-3)M均不能增强该通道的活性。百日咳毒素是已知的从相关G蛋白(G(I)或G(O))解偶联的M受体，在没有激动剂的情况下，它不能抑制ATP和镁离子诱导的K+通道的激活。在测试镁-三磷酸腺苷需求是否是强制性磷酸化反应的实验中，三磷酸腺苷被腺酰亚胺二磷酸(AMP-PNP)取代，腺苷二磷酸是三磷酸腺苷的一种类似物，它是一种抗水解性的三磷酸腺苷。当用AMP-PNP取代ATP时，这种K+通道活性不存在。这种依赖镁离子-三磷酸腺苷的磷酸化似乎不涉及蛋白激酶C的激活，因为蛋白激酶C的激活剂12-O-十四酰佛波醇-13-乙酸酯(TPA，10 nM)并不增加细胞贴附的膜片构型中这一M胆碱型K+通道的自发开放；该通道活性也不被1-(5-isoquinolinylsulphonyl)-2-methylpiperazine(H-7，20-mU-M)抑制。为了评估通道激活所需的磷酸化反应可能涉及膜结合的核苷酸二磷酸激酶(NDPK)的可能性，我们使用了已知能够替代ATP的核苷酸三磷酸作为NDPK的底物。与这种可能性一致的是，尿苷5‘-三磷酸(UTP)(1 MM)、胸苷(TTP)(1 MM)和鸟苷(GTP)(1 MM)在2 mM-MgCl2存在下均能激活这些K+通道，但GTP的作用远不如ATP.10。NDPK参与这些K+通道自发激活的其他证据是通过应用已知的该酶的非特异性抑制剂的核苷酸获得的。腺苷5‘-二磷酸(ADP，500mU-M)、鸟苷5’-O-(2-硫代二磷酸)(GDp-β-S，100mU-M)和鸟苷5‘-二磷酸(GDP25mU-M)分别强烈抑制ATP和Mg2+诱导的自发性钾通道活动。这些结果表明，即使在M受体不被激动剂占据的情况下，M受体也受细胞内核苷酸和镁离子的调节；这些结果表明，转磷酸化反应可能是这种调节所必需的，可能涉及到膜结合的核苷酸二磷酸激酶。
1. Intracellular mechanism(s) for controlling the opening of muscarinic K+ channels in the absence of an applied muscarinic agonist were studied in rabbit atrium by applying the patch clamp technique to isolate single myocytes.2. In the cell-attached patch configuration, currents due to the activity of both the muscarinic K+ channel and the inward rectifying K+ channel were recorded. However, while the inward rectifying K+ channel currents were observed in only ten patches of 211 examined, spontaneous opening (i.e. in the absence of a muscarinic agonist) of the muscarinic K+ channel currents was observed in all patches examined in these atrial cells.3. The single-channel currents due to spontaneous opening of muscarinic K+ channels were identified on the basis of their very similar conductance and gating properties to the unitary events which have been recorded when 0.5-mu-M-acetylcholine is included in the pipette and 10-mu-M-GTP is present in the internal side of the patch membrane.4. Although the spontaneous opening of the muscarine K+ channels disappeared soon after excision of the patch membrane, this type of channel activity reappeared following application of ATP and MgCl2 to the internal side of the torn-off patch, as expected from previous publications.5. The K+ channel activity induced by the ATP and Mg2+ (measured as the product of the number of channels, N, times the probability of opening, P(o)) was strongly dependent upon concentration of free Mg2+; it was half-maximal at 2.2 x 10(-4) M [Mg2+]i. However, after the muscarinic K+ channels had been activated by 100-mu-M-guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S) together with ATP and Mg2+, an increase in the Mg2+ concentration from 5.5 x 10(-5) to 2 x 10(-3) M failed to enhance this channel activity.6. Pertussis toxin, which is known to uncouple muscarinic receptors from associated G-proteins (G(i) or G(o)), failed to inhibit the ATP- and Mg2+ -induced activation of this K+ channel in the absence agonists.7. In experiments made to test whether the Mg2+-ATP requirements results from an obligatory phosphorylation reaction, ATP was replaced with adenylyl-imidodiphosphate (AMP-PNP), an analogue of ATP which is resistant to hydrolysis. This K+ channel activity was not present when ATP was replaced with AMP-PNP.8. This Mg2+ -ATP-dependent phosphorylation does not appear to involve activation of protein kinase C since 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 nM), an activator of protein kinase C, did not increase the spontaneous opening of this muscarinic K+ channel in the cell-attached patch configuration; and this channel activity was not inhibited by 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7, 20-mu-M).9. To assess the possibility that the phosphorylation reaction required for channel activation may involve a membrane-bound nucleotide diphosphate kinase (NDPK) we applied nucleotide triphosphates which are known to be able to substitute for ATP as substrates for the NDPK. Consistent with this possibility, uridine 5'-triphosphate (UTP) (1 mM), thymidine 5'-triphosphate (TTP) (1 mM) and guanosine 5'-triphosphate (GTP) (1 mM) were able to activate these K+ channels in the presence of 2 mM-MgCl2; however GTP was much less effective than ATP.10. Additional evidence for the involvement of NDPK in the spontaneous activation of these K+ channels was obtained by applying nucleotides known to be non-specific inhibitors of this enzyme. Adenosine 5'-diphosphate (ADP, 500-mu-M), guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S, 100-mu-M) and guanosine 5'-diphosphate (GDP, 25-mu-M) each strongly inhibited the spontaneous K+ channel activity induced by ATP and Mg2+.11. These results demonstrate that the muscarinic K+ channel is regulated by intracellular nucleotides and Mg2+, even when the muscarinic receptors are not occupied by agonist; and they suggest that a transphosphorylation reaction may be essential for this regulation, possibly involving a membrane-bound nucleotide diphosphate kinase.