Involvement of nonselective cation channels in the depolarisation initiating vasomotion.

1. Coordinated oscillations in diameter occur spontaneously in cerebral vessels and depend on the opening of voltage dependent calcium channels. However, the mechanism that induces the initial depolarisation has remained elusive. We investigated the involvement of canonical transient receptor potential (TRPC) channels, which encode nonselective cation channels passing Na(+) and Ca(2+) currents, by measuring changes in diameter, intracellular Ca(2+) and membrane potential in branches of juvenile rat basilar arteries. 2. Removal of extracellular Ca(2+) abolished vasomotion and relaxed arteries, but paradoxically produced depolarisation. 3. Decrease in temperature to 24 degrees C or inhibition of phospholipase C (PLC) abolished vasomotion, hyperpolarised and relaxed arteries and decreased intracellular Ca(2+). 4. Reduction in the driving force for Na(+) through decrease in extracellular Na(+) produced similar effects and prevented the depolarisation elicited by removal of extracellular Ca(2+). 5. Nonselective TRP channel blockers, SKF96365 and gadolinium, mimicked the effects of inhibition of the PLC pathway. 6. Depolarisation of vessels in which TRP channels were blocked with SKF96365 reinstated vascular tone and vasomotion. 7. Quantitative polymerase chain reaction revealed TRPC1 as the predominantly expressed TRPC subtype. 8. Incubation with a function blocking TRPC1 antibody delayed the onset of vasomotion. 9. We conclude that nonselective cation channels contribute to vasoconstriction and vasomotion of cerebral vessels by providing an Na(+)-induced depolarisation that activates voltage dependent calcium channels. Our antibody data suggest the involvement of TRPC1 channels that might provide a target for treatment of therapy-refractory vasospasm.

Non-L-type voltage-dependent calcium channels control vascular tone of the rat basilar artery.

1. Constriction of cerebral arteries is considered to depend on L-type voltage-dependent calcium channels (VDCCs); however, many previous studies have used antagonists with potential non-selective actions. Our aim was to determine the expression and function of VDCCs in the rat basilar artery. 2. The relative expression of VDCC subtypes was assessed using quantitative polymerase chain reaction and immunohistochemistry. Data were correlated with physiological studies of vascular function. Domains I-II of the T channel subtypes expressed in the rat basilar artery were cloned and sequenced. 3. Blockade of L-type channels with nifedipine had no effect on vascular tone. In contrast, in the presence of nifedipine, hyperpolarization of short arterial segments produced relaxation, whereas depolarization of quiescent segments evoked constriction. 4. The mRNA and protein for L- and T-type VDCCs were strongly expressed in the main basilar artery and side branches, with Ca(V)3.1 and Ca(V)1.2 the predominant subtypes. 5. T-Type VDCC blockers (i.e. 1 micromol/L mibefradil, 10 micromol/L pimozide and 100 micromol/L flunarizine) decreased intracellular calcium in smooth muscle cells, relaxed and hyperpolarized arteries, whereas nickel chloride (100 micromol/L) had no effect. In contrast with nifedipine, 10 micromol/L nimodipine produced hyperpolarization and relaxation. 6. When arteries were relaxed with 10 micromol/L U73122 (a phospholipase C inhibitor) in the presence of nifedipine, 40 mmol/L KCl evoked depolarization and constriction, which was significantly reduced by 1 micromol/L mibefradil. 7. Sequencing of domains I-II revealed splice variants of Ca(V)3.1, which may impact on channel activity. 8. We conclude that vascular tone of the rat basilar artery results from calcium influx through nifedipine-insensitive VDCCs with pharmacology consistent with Ca(V)3.1 T-type channels.

Alternative splicing within the I-II loop controls surface expression of T-type Ca(v)3.1 calcium channels.

Molecular diversity of T-type/Ca(v)3 Ca2+ channels is created by expression of three genes and alternative splicing of those genes. Prompted by the important role of the I-II linker in gating and surface expression of Ca(v)3 channels, we describe here the properties of a novel variant that partially deletes this loop. The variant is abundantly expressed in rat brain, even exceeding transcripts with the complete exon 8. Electrophysiological analysis of the Delta8b variant revealed enhanced current density compared to Ca(v)3.1a, but similar gating. Luminometry experiments revealed an increase in the expression of Delta8b channels at the plasma membrane. We conclude that alternative splicing of Ca(v)3 channels regulates surface expression and may underlie disease states in which T-channel current density is increased.

CADASIL mutations impair Notch3 glycosylation by Fringe.

Mutations in the NOTCH3 gene trigger adult-onset stroke and vascular dementia in patients with CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). All CADASIL mutations described to date affect the epidermal growth factor-like (EGF-like) repeats located in the extracellular domain of the Notch3 receptor. These domains are also the target of sequential complex O-linked glycosylation mediated by protein O-fucosyltransferase 1 and Fringe. We investigated whether O-fucosylation or Fringe-mediated elongation of O-fucose on Notch3 is impaired by CADASIL mutations. Biochemical studies of a Notch3 fragment containing the first five EGF-like repeats of Notch3, including the mutational hot spot, showed that CADASIL mutations do not affect the addition of O-fucose but do impair carbohydrate chain elongation by Fringe. CADASIL changes also induced aberrant homodimerization of mutant Notch3 fragments and heterodimerization of mutant Notch3 with Lunatic Fringe itself. Together, these data suggest that Fringe plays a role in CADASIL pathophysiology.