Garza-López Edgar, Sandoval Alejandro, González-Ramírez Ricardo, Gandini María A, Van den Maagdenberg Arn, De Waard Michel, Felix Ricardo
Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute, Mexico City, Mexico.
Biochim Biophys Acta. 2012 Aug;1822(8):1238-46. doi: 10.1016/j.bbadis.2012.04.008. Epub 2012 Apr 20.
Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca(V)2.1 voltage-gated Ca(2+) channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca(V)2.1α(1) subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK-293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca(V)2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca(2+) channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.
1型家族性偏瘫性偏头痛（FHM-1）是一种伴有先兆的偏头痛单基因形式，其特征为典型偏头痛性头痛反复发作，在先兆期伴有短暂性偏瘫。在一部分患者中，癫痫和小脑共济失调等其他症状也是临床表型的一部分。FHM-1由CACNA1A基因突变引起，该基因编码Ca（V）2.1电压门控Ca（2+）通道的孔形成亚基。尽管越来越多的FHM-1突变的功能效应已得到表征，但对于这些突变中的大多数对通道功能的G蛋白调节的影响仍缺乏了解。在这里，我们探讨了G蛋白依赖性调节对分别导致有和没有小脑共济失调的FHM-1的W1684R和V1696I突变的影响。将这两种突变引入人Ca（V）2.1α（1）亚基，并在人胚肾293（HEK-293）细胞中进行异源表达后，使用膜片钳记录研究其功能后果。当与人类μ-阿片受体共表达时，激动剂[d-Ala2，N-MePhe4，Gly-ol]-脑啡肽（DAMGO）的应用抑制了通过野生型（WT）和突变型Ca（V）2.1通道的电流，这与G蛋白偶联受体对这些通道的已知调节一致。预脉冲易化是表征直接电压依赖性G蛋白调节缓解的一种方式，两种FHM-1突变均使其降低。此外，对易化起始和衰减的动力学分析表明，W1684R和V1696I突变影响Gβγ二聚体从通道复合物的表观解离和重新结合速率，表明突变可能改变G蛋白-Ca（2+）通道亲和力。这些生物物理研究可能为FHM-1的病理生理学提供新的线索。
