School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AJ, UK.
J Physiol. 2011 Jul 1;589(Pt 13):3085-101. doi: 10.1113/jphysiol.2010.204735. Epub 2011 Apr 26.
Modulation of presynaptic voltage-dependent Ca2+ channels is a major means of controlling neurotransmitter release. The CaV2.2Ca2+ channel subunit contains several inhibitory interaction sites for Gβγ subunits, including the amino terminal (NT) and I-II loop. The NT and I-II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV2 channel activity. Here, we investigate the effects of an amino terminal (CaV2.2[45-55]) 'NT peptide' and a I-II loop alpha interaction domain (CaV2.2[377-393]) 'AID peptide' on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV2.2 amino terminal and I-II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation.
突触前电压依赖性 Ca2+ 通道的调节是控制神经递质释放的主要手段。CaV2.2Ca2+ 通道亚基包含几个 Gβγ 亚基的抑制性相互作用位点，包括氨基末端（NT）和 I-II 环。NT 和 I-II 环也被提议发生 G 蛋白门控抑制性相互作用，而 NT 本身也被提议抑制 CaV2 通道活性。在这里，我们研究了氨基末端（CaV2.2[45-55]）“NT 肽”和 I-II 环α相互作用域（CaV2.2[377-393]）“AID 肽”对颈上交感神经节神经元（SCGN）突触传递、Ca2+通道活性和 G 蛋白调节的影响。将 NT 或 AID 肽注入 SCGN 突触前，抑制突触传递，并减弱去甲肾上腺素诱导的 G 蛋白调节。在分离的 SCGN 中，NT 和 AID 肽减少全细胞 Ca2+电流幅度，改变 Ca2+通道激活的电压依赖性，并减弱去甲肾上腺素诱导的 G 蛋白调节。NT 和 AID 肽的共同应用否定了抑制作用。总之，这些数据支持肽与突触前 Ca2+通道的直接相互作用，对电流幅度和门控的影响代表了抑制突触传递的可能机制。NT 肽中报道的决定 Ca2+通道功能的残基突变否定了对突触传递、Ca2+电流幅度和门控以及 G 蛋白调节的抑制作用。在推测的 G 蛋白调节的 QXXER 基序内的突变不消除 AID 肽的抑制作用。这项研究表明，CaV2.2 的氨基末端和 I-II 环为 Ca2+通道功能提供了分子决定因素；数据支持肽与 Ca2+通道的直接相互作用，以抑制突触传递并减弱 G 蛋白调节。
