Linn C L
Louisiana State University Health Sciences Center, Department of Cell Biology and Anatomy, New Orleans 70112, USA.
Vis Neurosci. 2000 May-Jun;17(3):473-82. doi: 10.1017/s0952523800173134.
Catfish cone horizontal cells contain a voltage-gated L-type calcium channel that is modulated by activation of metabotropic glutamate receptors (mGluRs). Activation of group I mGluRs with the mGluR I agonist, (S)-3,5-dihydroxyphenylglycine [(S) 3,5-DHPG], potentiated peak calcium current amplitude, shifted the membrane potential corresponding to peak current activity, and widened the calcium current's activation range. In this study, we have examined the mechanisms linking activation of the mGluRs with "up-regulation" of calcium current activity. Under whole-cell voltage-clamp conditions favoring expression of the L-type calcium current, we provide evidence that activation of mGluRs initiate the diacylglyceral (DG) second messenger pathway to activate protein kinase C (PKC) and up-regulate calcium channel activity. This evidence was based on results using a number of PKC activators and inhibitors. PKC activators mimicked the effect of (S) 3,5-DHPG on calcium current activity. Up-regulation of the calcium channel by PKC activators or (S) 3,5-DHPG was eliminated if PKC inhibitors were present. These results also demonstrated that activation of group I mGluRs were linked to a pertussis toxin sensitive G-protein. When the GTP analog, guanosine 5-0-(3-thiotriphosphate (GTPgammaS), was allowed to diffuse into voltage-clamp cells, up-regulation of the calcium channel occurred and mimicked the effect of (S) 3,5-DHPG. However, when pertussis toxin (PTX) was allowed to diffuse into the cell along with GTPgammaS, GTPgammaS failed to modulate calcium current activity. IP3 (inositol 1,4,5 triphosphate) is a second product produced by activation of group I mGluRs. Once formed, IP3 can trigger calcium release from IP3-sensitive intracellular stores. To determine if the IP3 second messenger system was involved in up-regulation of calcium channel, (S) 3,5-DHPG was applied to voltage-clamped cone horizontal cells containing different concentrations of the calcium buffer, EGTA. Low concentrations of EGTA failed to buffer calcium released from intracellular stores. In the presence of low EGTA concentrations, (S) 3,5-DHPG's enhancement of the calcium current amplitude was reduced. Inhibition of the calcium current amplitude in low concentrations of EGTA was eliminated in the presence of the intracellular calcium store blocker, heparin. These results suggest that both the DG and IP3 second messenger pathways are involved in modulation of the voltage-gated calcium channel in catfish cone horizontal cells. The DG pathway up-regulates the voltage-gated calcium channel activity whereas calcium released from IP3 intracellular stores inhibits peak current amplitude.
鲶鱼视锥水平细胞含有一种电压门控L型钙通道，该通道受代谢型谷氨酸受体（mGluRs）激活的调节。用mGluR I激动剂（S）-3,5-二羟基苯甘氨酸[（S）3,5-DHPG]激活I组mGluRs，可增强钙电流峰值幅度，使对应于峰值电流活动的膜电位发生偏移，并拓宽钙电流的激活范围。在本研究中，我们研究了将mGluRs激活与钙电流活动“上调”联系起来的机制。在有利于L型钙电流表达的全细胞电压钳制条件下，我们提供证据表明，mGluRs的激活启动了二酰甘油（DG）第二信使途径，以激活蛋白激酶C（PKC）并上调钙通道活性。这一证据基于使用多种PKC激活剂和抑制剂的结果。PKC激活剂模拟了（S）3,5-DHPG对钙电流活动的影响。如果存在PKC抑制剂，PKC激活剂或（S）3,5-DHPG对钙通道的上调作用就会消除。这些结果还表明，I组mGluRs的激活与一种对百日咳毒素敏感的G蛋白有关。当鸟苷5'-O-（3-硫代三磷酸）（GTPγS）这种GTP类似物扩散到电压钳制细胞中时，钙通道发生上调并模拟了（S）3,5-DHPG的作用。然而，当百日咳毒素（PTX）与GTPγS一起扩散到细胞中时，GTPγS无法调节钙电流活动。IP3（肌醇1,4,5-三磷酸）是I组mGluRs激活产生的另一种产物。一旦形成，IP3可触发从IP3敏感的细胞内储存库中释放钙。为了确定IP3第二信使系统是否参与钙通道的上调，将（S）3,5-DHPG应用于含有不同浓度钙缓冲剂乙二醇双四乙酸（EGTA）的电压钳制视锥水平细胞。低浓度的EGTA无法缓冲从细胞内储存库中释放的钙。在低EGTA浓度存在的情况下，（S）3,5-DHPG对钙电流幅度的增强作用减弱。在细胞内钙储存库阻滞剂肝素存在的情况下，低浓度EGTA中钙电流幅度的抑制作用消除。这些结果表明，DG和IP3第二信使途径都参与了鲶鱼视锥水平细胞中电压门控钙通道的调节。DG途径上调电压门控钙通道活性，而从IP3细胞内储存库中释放出钙则抑制峰值电流幅度。
