1. G protein-regulated inward rectifier K¤ (GIRK) channels were over-expressed in dissociated rat superior cervical sympathetic (SCG) neurones by co-transfecting green fluorescent protein (GFP)-, GIRK1-and GIRK2-expressing plasmids using the biolistic technique. Membrane currents were subsequently recorded with whole-cell patch electrodes. 2. Co-transfected cells had larger Ba¥-sensitive inwardly rectifying currents and 13 mV more negative resting potentials (in 3 mÒ [K¤]o) than non-transfected cells, or cells transfected with GIRK1 or GIRK2 alone. 3. Carbachol (CCh, 1-30 ìÒ) increased the inwardly rectifying current in 70% of GIRK1+ GIRK2-transfected cells by 261 ± 53 % (n = 6, CCh 30 ìÒ) at −120 mV, but had no effect in non-transfected cells or in cells transfected with GIRK1 or GIRK2 alone. Pertussis toxin prevented the effect of carbachol but had no effect on basal currents. 4. The effect of CCh was antagonized by 6 nÒ tripitramine but not by 100 nÒ pirenzepine, consistent with activation of endogenous Mµ muscarinic acetylcholine receptors. 5. In contrast, inhibition of the voltage-activated Ca¥ current by CCh was antagonized by 100 nÒ pirenzepine but not by 6 nÒ tripitramine, indicating that it was mediated by MÚ muscarinic acetylcholine receptors. 6. We conclude that endogenous Mµ and MÚ muscarinic receptors selectively couple to GIRK currents and Ca¥ currents respectively, with negligible cross-talk.8938

The transient receptor potential (TRP) superfamily of non-selective cationic channels are involved in several processes plausibly relevant to migraine pathophysiology, including multimodal sensory and pain perception, central and peripheral sensitization, and regulation of calcium homeostasis. With the aim of identifying single nucleotide polymorphisms (SNPs) in TRP genes that may confer increased genetic susceptibility to migraine, we carried out a case-control genetic association study with replication, including a total of 1,040 cases and 1,037 controls. We genotyped 149 SNPs covering 14 TRP genes with known brain expression. The two-stage study comprised samples of 555 and 485 Spanish, Caucasian patients, selected according to the ICHD-II criteria for the diagnosis of migraine without aura (MO) or migraine with aura (MA). In the discovery sample, 19 SNPs in ten TRP genes showed nominal association (P < 0.05) with MO, MA, or overall migraine. In the replication sample, nominal association was confirmed for TRPV3 rs7217270 in MA and TRPV1 rs222741 in the overall migraine group. Risk haplotypes were identified for seven of the genes showing nominal association in the discovery set, but none of them was replicated. The present findings suggest that members of the vanilloid TRPV subfamily of receptors contribute to the genetic susceptibility to migraine in the Spanish population.

Rat superior cervical ganglion (SCG) neurons express lowthreshold noninactivating M-type potassium channels (I K(M)), which can be inhibited by activation of M 1 muscarinic receptors (M 1 mAChR) and bradykinin (BK) B 2 receptors. Inhibition by the M 1 mAChR agonist oxotremorine methiodide (Oxo-M) is mediated, at least in part, by the pertussis toxin-insensitive G-protein G␣ q (Caulfield et al., 1994; Haley et al., 1998a), whereas BK inhibition involves G␣ q and/or G␣ 11 (Jones et al., 1995). G␣ q and G␣ 11 can stimulate phospholipase C-␤ (PLC-␤), raising the possibility that PLC is involved in I K(M) inhibition by Oxo-M and BK. RT-PCR and antibody staining confirmed the presence of PLC-␤1,-␤2,-␤3, and-␤4 in rat SCG. We have tested the role of two PLC isoforms (PLC-␤1 and PLC-␤4) using antisense-expression constructs. Antisense constructs, consisting of the cytomegalovirus promoter driving antisense cRNA corresponding to the 3Ј-untranslated regions of PLC-␤1 and PLC-␤4, were injected into the nucleus of dissociated SCG neurons. Injected cells showed reduced antibody staining for the relevant PLC-␤ isoform when compared to uninjected cells 48 hr later. BK inhibition of I K(M) was significantly reduced 48 hr after injection of the PLC-␤4, but not the PLC-␤1, antisenseencoding plasmid. Neither PLC-␤ antisense altered M 1 mAChR inhibition by Oxo-M. These data support the conclusion of Cruzblanca et al. (1998) that BK, but not M 1 mAChR, inhibition of I K(M) involves PLC and extends this finding by indicating that PLC-␤4 is involved.

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