Alternative splicing is known to generate multiple functionally distinct calcium channel variants that exhibit unique spatial and temporal expression patterns. In humans, naturally occurring mutations in genes encoding calcium channel pore forming α 1 -subunits are associated with several severe hereditary disorders although it remains to be described whether there exists any relationship between the physiological effects of these mutations and calcium channel splice variation. In the present study, we systematically compare the biophysical effects of three type-1 familial hemiplegic migraine (FHM-1) mutations in two predominant splice variants of the neuronal Ca V 2.1 P/Q-type channel. All three FHM-1 mutations cause a greater hyperpolarizing shift in voltage-dependent properties when expressed in the short carboxyl terminus variant (Ca V 2.1 Δ47) compared to the long variant (Ca V 2.1 +47). Furthermore, the FHM-1 mutations also exhibit differential splice variant-specific effects on recovery from inactivation and accumulation of inactivation during tonic and burst firing. Our findings provide important insight concerning the role of calcium channel alternatively spliced variants and the molecular pathophysiology of FHM-1 and potentially of other calcium channelopathies.

The dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals. The enzyme diacylglycerol kinase ζ (DGKζ) serves as a scaffold to assemble a signaling complex that functions as a RhoA-specific RhoGDI dissociation factor. DGKζ deficiency impairs RhoA activation and stress fiber formation in fibroblasts.

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca 2؉ channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca 2؉ channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (␣ 1G ), Cav3.2 (␣ 1H ), and Cav3.3 (␣ 1I ) T-type Ca 2؉ channels were co-expressed with the M1 G␣ q/11 -coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca 2؉ currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca 2؉ channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of G␣ and G␤␥ proteins and gain-of-function experiments with genetically encoded G␣ subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through G␣ q/11 . This is supported by experiments showing that activation of the M3 and M5 G␣ q/11 -coupled mAChRs also causes inhibition of Cav3.3 currents, although G␣ i -coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation.

BackgroundUnraveling the signaling pathways responsible for the establishment of a metastatic phenotype in carcinoma cells is critically important for understanding the pathology of cancer. The acquisition of cell motility is a key property of metastatic tumor cells and is a prerequisite for invasion. Rho GTPases regulate actin cytoskeleton reorganization and the cellular responses required for cell motility and invasion. Diacylglycerol kinase ζ (DGKζ), an enzyme that phosphorylates diacylglycerol to yield phosphatidic acid, regulates the activity of the Rho GTPases Rac1 and RhoA. DGKζ mRNA is highly expressed in several different colon cancer cell lines, as well as in colon cancer tissue relative to normal colonic epithelium, and thus may contribute to the metastatic process.MethodsTo investigate potential roles of DGKζ in cancer metastasis, a cellular, isogenic model of human colorectal cancer metastatic transition was used. DGKζ protein levels, Rac1 and RhoA activity, and PAK phosphorylation were measured in the non-metastatic SW480 adenocarcinoma cell line and its highly metastatic variant, the SW620 line. The effect of DGKζ silencing on Rho GTPase activity and invasion through Matrigel-coated Transwell inserts was studied in SW620 cells. Invasiveness was also measured in PC-3 prostate cancer and MDA-MB-231 breast cancer cells depleted of DGKζ.ResultsDGKζ protein levels were elevated approximately 3-fold in SW620 cells compared to SW480 cells. There was a concomitant increase in active Rac1 in SW620 cells, as well as substantial increases in the expression and phosphorylation of the Rac1 effector PAK1. Similarly, RhoA activity and expression were increased in SW620 cells. Knockdown of DGKζ expression in SW620 cells by shRNA-mediated silencing significantly reduced Rac1 and RhoA activity and attenuated the invasiveness of SW620 cells in vitro. DGKζ silencing in highly metastatic MDA-MB-231 breast cancer cells and PC-3 prostate cancer cells also significantly attenuated their invasiveness.ConclusionElevated DGKζ expression contributes to increased Rho GTPase activation and the enhanced motility of metastatic cancer cells. These findings warrant further investigation of the clinical relevance of DGKζ upregulation in colon and other cancers. Interfering with DGKζ function could provide a means of inhibiting invasion and metastasis.