McCool B A, Pin J P, Harpold M M, Brust P F, Stauderman K A, Lovinger D M
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37203, USA.
J Neurophysiol. 1998 Jan;79(1):379-91. doi: 10.1152/jn.1998.79.1.379.
We have shown previously that metabotropic glutamate receptors with group I-like pharmacology couple to N-type and P/Q-type calcium channels in acutely isolated cortical neurons using G proteins most likely belonging to the Gi/Go subclass. To better understand the potential mechanisms forming the basis for group I mGluR modulation of voltage-gated calcium channels in the CNS, we have examined the ability of specific mGluRs to couple to neuronal N-type (alpha1B-1/alpha2delta/beta1b) and P/Q-type (alpha1A-2/alpha2delta/beta1b) voltage-gated calcium channels in an HEK 293 heterologous expression system. Using the whole cell patch-clamp technique where intracellular calcium is buffered to low levels, we have shown that group I receptors inhibit both N-type and P/Q-type calcium channels in a voltage-dependent fashion. Similar to our observations in cortical neurons, this voltage-dependent inhibition is mediated almost entirely by N-ethylmaleimide (NEM)-sensitive heterotrimeric G proteins, strongly suggesting that these receptors can use Gi/Go-like G proteins to couple to N-type and P/Q-type calcium channels. However, inconsistent with the apparent NEM sensitivity of group I modulation of calcium channels, modulation of N-type channels in group I mGluR-expressing cells was only partially sensitive to pertussis toxin (PTX), indicating the potential involvement of both PTX-sensitive and -resistant G proteins. The PTX-resistant modulation was voltage dependent and entirely resistant to NEM and cholera toxin. A time course of treatment with PTX revealed that this toxin caused group I receptors to slowly shift from using a primarily NEM-sensitive G protein to using a NEM-resistant form. The PTX-induced switch from NEM-sensitive to -resistant modulation was also dependent on protein synthesis, indicating some reliance on active cellular processes. In addition to these voltage-dependent pathways, perforated patch recordings on group I mGluR-expressing cells indicate that another slowly developing, calcium-dependent form of modulation for N-type channels may be seen when intracellular calcium is not highly buffered. We conclude that group I mGluRs can modulate neuronal Ca2+ channels using a variety of signal transduction pathways and propose that the relative contributions of different pathways may exemplify the diversity of responses mediated by these receptors in the CNS.
我们之前已经表明，具有I组样药理学特性的代谢型谷氨酸受体，在急性分离的皮层神经元中，通过很可能属于Gi/Go亚类的G蛋白，与N型和P/Q型钙通道偶联。为了更好地理解构成中枢神经系统中I组代谢型谷氨酸受体对电压门控钙通道调节基础的潜在机制，我们在HEK 293异源表达系统中研究了特定代谢型谷氨酸受体与神经元N型（α1B-1/α2δ/β1b）和P/Q型（α1A-2/α2δ/β1b）电压门控钙通道偶联的能力。使用全细胞膜片钳技术，将细胞内钙缓冲至低水平，我们已经表明I组受体以电压依赖性方式抑制N型和P/Q型钙通道。与我们在皮层神经元中的观察结果相似，这种电压依赖性抑制几乎完全由对N-乙基马来酰亚胺（NEM）敏感的异源三聚体G蛋白介导，强烈表明这些受体可以使用类似Gi/Go的G蛋白与N型和P/Q型钙通道偶联。然而，与I组对钙通道调节明显的NEM敏感性不一致的是，在表达I组代谢型谷氨酸受体的细胞中，N型通道的调节仅部分对百日咳毒素（PTX）敏感，表明PTX敏感和抗性G蛋白都可能参与其中。PTX抗性调节是电压依赖性的，并且完全抵抗NEM和霍乱毒素。用PTX处理的时间进程表明，这种毒素导致I组受体从主要使用对NEM敏感的G蛋白缓慢转变为使用对NEM抗性的形式。PTX诱导的从对NEM敏感到抗性调节的转变也依赖于蛋白质合成，表明对活跃的细胞过程有一定依赖性。除了这些电压依赖性途径外，对表达I组代谢型谷氨酸受体的细胞进行穿孔膜片钳记录表明，当细胞内钙没有被高度缓冲时，对于N型通道可能会出现另一种缓慢发展的、钙依赖性的调节形式。我们得出结论，I组代谢型谷氨酸受体可以使用多种信号转导途径调节神经元Ca2+通道，并提出不同途径的相对贡献可能体现了这些受体在中枢神经系统中介导的反应的多样性。
