Keum Dongil, Baek Christina, Kim Dong-Il, Kweon Hae-Jin, Suh Byung-Chang
Department of Brain Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 711-873, South Korea.
Department of Brain Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 711-873, South Korea
J Gen Physiol. 2014 Oct;144(4):297-309. doi: 10.1085/jgp.201411245. Epub 2014 Sep 15.
G protein-coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca(2+), and phosphatidylinositol 4,5-bisphosphate (PIP2), to modulate N-type voltage-gated Ca(2+) (CaV2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which GqPCRs inhibit CaV2.2 channel current are not completely understood. Here, we report that the location of CaV β subunits is key to determining the voltage dependence of CaV2.2 channel modulation by GqPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M1 receptors, together with CaV N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for CaV2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of CaV2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of CaV2.2 current after M1 receptor activation was markedly reduced and delayed, whereas the delay between PIP2 hydrolysis and inhibition of CaV2.2 current was decreased. When the Gβγ-insensitive CaV2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M1 receptors act through Gβγ to inhibit CaV2.2 channels bearing membrane-localized CaV β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M1 muscarinic inhibition of CaV2.2 channels, whereas it increased inhibition mediated by PIP2 depletion. Together, our results indicate that, with membrane-localized CaV β subunits, CaV2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP2-mediated voltage-independent regulation. Thus, the voltage dependence of GqPCR regulation of calcium channels can be determined by the location of isotype-specific CaV β subunits.
G蛋白偶联受体（GPCRs）通过分子信使（如Gβγ、Ca²⁺和磷脂酰肌醇4,5-二磷酸（PIP2））发出信号，以调节N型电压门控Ca²⁺（CaV2.2）通道，在调节突触传递中起关键作用。然而，GqPCRs抑制CaV2.2通道电流的细胞途径尚未完全明确。在此，我们报告CaVβ亚基的位置是决定GqPCRs对CaV2.2通道调节的电压依赖性的关键。将毒蕈碱激动剂氧化震颤素-M应用于表达M1受体的tsA-201细胞，同时表达CaV N型α1B、α2δ1和膜定位的β2a亚基，使CaV2.2激活的电流-电压关系向右移动5 mV，并减缓电流激活。强去极化预脉冲可解除毒蕈碱对CaV2.2活性的抑制。此外，当β-肾上腺素能受体激酶（与Gβγ结合）的C末端与N型通道共表达时，M1受体激活后对CaV2.2电流的抑制明显降低且延迟，而PIP2水解与CaV2.2电流抑制之间的延迟减少。当表达对Gβγ不敏感的CaV2.2 α1C-1B嵌合体时，钙电流的电压依赖性抑制几乎消失，表明M1受体通过Gβγ作用于抑制带有膜定位CaVβ2a亚基的CaV2.2通道。胞质β亚基如β2b和β3以及棕榈酰化阴性突变体β2a（C3,4S）的表达降低了M1毒蕈碱对CaV2.2通道抑制的电压依赖性，而增加了由PIP2耗竭介导的抑制。总之，我们的结果表明，对于膜定位的CaVβ亚基，CaV2.2通道受到Gβγ介导的电压依赖性抑制，而胞质定位的β亚基赋予更有效的PIP2介导的电压非依赖性调节。因此，GqPCR对钙通道调节的电压依赖性可由同种型特异性CaVβ亚基的位置决定。
