Dramatically different levels of Cacna1a gene expression between pre-weaning wild type and leaner mice.

Loss of function mutations of the CACNA1A gene, coding for the α1A subunit of P/Q type voltage-gated calcium channel (Ca(V)2.1), are responsible for Episodic Ataxia type 2 (EA2), an autosomal dominant disorder. A dominant negative effect of the EA2 mutated protein, rather than a haploinsufficiency mechanism, has been hypothesised both for protein-truncating and missense mutations. We analysed the cacna1a mRNA expression in leaner mice carrying a cacna1a mutation leading to a premature stop codon. The results showed a very low mutant mRNA expression compared to the wild type allele. Although the mutant mRNA slightly increases with age, its low level is likely due to degradation by nonsense mediated decay, a quality control mechanism that selectively degrades mRNA harbouring premature stop codons. These data have implications for EA2 in humans, suggesting a haploinsufficiency mechanism at least for some of the CACNA1A mutations leading to a premature stop codon.

Severe and progressive neurotransmitter release aberrations in familial hemiplegic migraine type 1 Cacna1a S218L knock-in mice.

Familial hemiplegic migraine type 1 (FHM1) is caused by mutations in the CACNA1A gene, encoding neuronal presynaptic Ca(V)2.1 (P/Q-type) Ca(2+) channels. These channels mediate neurotransmitter release at many central synapses and at the neuromuscular junction (NMJ). Mutation S218L causes a severe neurological phenotype of FHM and, additionally, ataxia and susceptibility to seizures, delayed brain edema, and fatal coma after minor head trauma. Recently, we generated a Cacna1a S218L knock-in mutant mouse, displaying these features and reduced survival. A first electrophysiological study showed high susceptibility for cortical spreading depression, enhanced neuronal soma Ca(2+) influx, and at diaphragm NMJs, a considerable increase of neurotransmitter release. We here assessed the function of S218L knock-in NMJs at several muscle types in great detail. Pharmacological analyses using specific Ca(V) subtype-blocking toxins excluded compensatory contribution of non-Ca(V)2.1 channels. Endplate potentials were considerably broadened at many NMJs. High rate (40 Hz)-evoked acetylcholine release was slightly reduced; however, it was not associated with block of neurotransmission causing weakness, as assessed with grip strength measurements and in vitro muscle contraction experiments. The synaptopathy clearly progressed with age, including development of an increased acetylcholine release at low-rate nerve stimulation at physiological extracellular Ca(2+) concentration and further endplate potential broadening. Our results suggest enhanced Ca(2+) influx into motor nerve terminals through S218L-mutated presynaptic Ca(V)2.1 channels, likely because of the earlier reported negative shift of activation potential and reduced inactivation. Similar severe aberrations at central synapses of S218L mutant mice and humans may underlie or contribute to the drastic neurological phenotype.

Different relationship of N- and P/Q-type Ca2+ channels to channel-interacting slots in controlling neurotransmission at cultured hippocampal synapses.

Synaptic transmission at CNS synapses is often mediated by joint actions of multiple Ca(2+) channel subtypes, most prominently, P/Q- and N-type. We have proposed that P/Q-type Ca(2+) channels saturate type-preferring slots at presynaptic terminals, which impose a ceiling on the synaptic efficacy of the channels. To test for analogous interactions for presynaptic N-type Ca(2+) channels, we overexpressed their pore-forming Ca(V)2.2 subunit in cultured mouse hippocampal neurons, recorded excitatory synaptic transmission from transfected cells, and dissected the contributions of N-, P/Q-, and R-type channels with subtype-specific blockers. Overexpression of Ca(V)2.2 did not increase the absolute size of the EPSC even though somatic N-type current was augmented by severalfold. Thus, the strength of neurotransmission is saturated with regard to levels of Ca(2+) channel expression for both N-type and P/Q-type channels. Overexpression of Ca(2+)-impermeable Ca(V)2.2 subunits decreased EPSC size, corroborating competition for channel slots. Striking asymmetries between N- and P/Q-type channels emerged when their relative contributions were compared with channel overexpression. Overexpressed N-type channels could competitively displace P/Q-type channels from P/Q-preferring slots and take over the role of supporting transmission. The converse was not found with overexpression of P/Q-type channels, regardless of their C-terminal domain. We interpret these findings in terms of two different kinds of presynaptic slots at excitatory synapses, one accepting N-type channels but rejecting P/Q-type (N(specific)) and the other preferring P/Q-type but also accepting N-type (PQ(preferring)). The interaction between channels and slots governs the respective contributions of multiple channel types to neurotransmission and, in turn, the ability of transmission to respond to various stimulus patterns and neuromodulators.

Molecular and immunohistochemical studies in expression of voltage-dependent Ca2+ channels in dorsal root ganglia from streptozotocin-induced diabetic mice.

We have recently demonstrated that intrathecal injection of a selective P/Q-type blocker of the voltage-dependent Ca(2+) channels (VDCCs) significantly inhibited the mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice, its antinociceptive effect being greater than in controls. In this study, to further clarify the underlying mechanism of the STZ-induced hyperalgesia, we investigated the expression level of the VDCC alpha1A and alpha1B subunits in the dorsal root ganglia (DRGs) and the dorsal spinal cord under this hyperalgesia. Real-time PCR analysis showed mRNA expression of alpha1A (P/Q-type), but not alpha1B (N-type), was significantly increased in the DRGs from the STZ-induced diabetic mice. On the other hand, gene expression of both alpha1 subunits was not changed in the dorsal part of the spinal cord. In diabetic DRG neurons, the number of large nerve cells was significantly reduced, whereas small neurons were significantly increased. Immunohistochemical study demonstrated the alpha1A-positive neurons, but not alpha1B-positive neurons, increased significantly greater in diabetic DRGs than in control in all cell size. These results indicate that an alteration in expression of P/Q-type VDCCs, especially in the small and medium-diameter primary afferent fibers, in pain pathways ascending input to the spinal cord may be involved in hypersensitivity in STZ-induced diabetes.

Genetic background influences P/Q-type Ca2+ channel alpha1A subunit mRNA expression in olfactory bulb and reproductive ability of N-type Ca2+ channel alpha1B subunit-deficient mice.

The Ca2+ channel alpha1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although the N-type Ca2+ channel plays a role in a variety of neuronal functions, alpha1B-deficient mice did not show apparent behavioral abnormality. In a previous study, we observed a compensatory increase of mRNA expression of the P/Q-type Ca2+ channel alpha1A subunit gene in olfactory bulb of alpha1B-deficient mice with a CBA x C57BL/6 background; these mice showed a normal reproductive ability. In this study, we found that the mRNA expression level of the alpha1A subunit was the same in olfactory bulb of wild, heterozygous, and homozygous alpha1B-deficient mice with a CBA/JN background, and the homozygous male mice produced no offspring. These results suggest that the genetic background influences alpha1A subunit mRNA expression and reproductive ability in alpha1B-deficient mice.

A comparison of Ca2+ channel blocking mode between gabapentin and verapamil: implication for protection against hypoxic injury in rat cerebrocortical slices.

1 The mode of Ca(2+) channel blocking by gabapentin [1-(aminomethyl)cyclohexane acetic acid] was compared to those of other Ca(2+) channel blockers, and the potential role of Ca(2+) channel antagonists in providing protection against hypoxic injury was subsequently investigated in rat cerebrocortical slices. 2 mRNA for the alpha(2)delta subunits of Ca(2+) channels was found in rat cerebral cortex. 3 Nitric oxide (NO) synthesis estimated from cGMP formation was enhanced by KCl stimulation, which was mediated primarily by the activation of N- and P/Q-type Ca(2+) channels. Gabapentin blocked both types of Ca(2+) channels, and preferentially reversed the response to 30 mM K(+) stimulation compared with 50 mM K(+) stimulation. In contrast, verapamil preferentially inhibited the response to depolarization by the higher concentration (50 mM) of K(+). 4 Gabapentin inhibited KCl-induced elevation of intracellular Ca(2+) in primary neuronal culture. 5 Hypoxic injury was induced in cerebrocortical slices by oxygen deprivation in the absence (severe injury) or presence of 3 mM glucose (mild injury). Gabapentin preferentially inhibited mild injury, while verapamil suppressed only severe injury. omega-Conotoxin GVIA (omega-CTX) and omega-agatoxin IVA (omega-Aga) were effective in both models. 6 NO synthesis was enhanced in a manner dependent on the severity of hypoxic insults. Gabapentin reversed the NO synthesis induced by mild insults, while verapamil inhibited that elicited by severe insults. omega-CTX and omega-Aga were effective in both the cases. 7 Therefore, the data suggest that gabapentin and verapamil cause activity-dependent Ca(2+) channel blocking by different mechanisms, which are associated with their cerebroprotective actions and are dependent on the severity of hypoxic insults.

Neurotransmitter release from tottering mice nerve terminals with reduced expression of mutated P- and Q-type Ca2+-channels.

Neurotransmitter release is triggered by Ca2+-influx through multiple sub-types of high voltage-activated Ca2+-channels. Tottering mice have a mutation in the alpha1A pore-forming subunit of P- and Q-type Ca2+-channels, two prominent sub-types that regulate transmitter release from central nerve terminals. Immunoblotting analysis of purified forebrain terminals from tottering mice revealed an 85% reduction in the protein expression level of the mutated alpha1A subunit compared to expression of the alpha1A subunit in wild-type terminals. In contrast, the expression of the alpha1B subunit of the N-type Ca2+-channels was unchanged. Release of the amino acids glutamate and GABA and of the neuropeptide cholecystokinin (CCK) induced by a short (100 ms) depolarization pulse was unchanged in the terminals of tottering mice. Studies using specific blockers of Ca2+-channels however, revealed a reduced contribution of P- and Q-type Ca2+-channels to glutamate and cholecystokinin release, whereas a greater reliance on N-type Ca2+-channels for release of these transmitters was observed. In contrast, the contribution of the P-, Q- and N-type Ca2+-channels to the release of GABA was not altered in tottering mice. These results indicate that the expression of the alpha1A subunit was decreased in terminals from tottering mice, and that a decreased contribution of P- and Q-type Ca2+-channels to the release of glutamate and cholecystokinin was functionally compensated by an increased contribution of N-type Ca2+-channels.

Functional expression of L-, N-, P/Q-, and R-type calcium channels in the human NT2-N cell line.

The biophysical and pharmacological properties of voltage-gated calcium channel currents in the human teratocarcinoma cell line NT2-N were studied using the whole cell patch-clamp technique. When held at -80 mV, barium currents (I(Ba)s) were evoked by voltage commands to above -35 mV that peaked at +5 mV. When holding potentials were reduced to -20 mV or 5 mM barium was substituted for 5 mM calcium, there was a reduction in peak currents and a right shift in the current-voltage curve. A steady-state inactivation curve for I(Ba) was fit with a Boltzmann curve (V(1/2) = -43.3 mV; slope = -17.7 mV). Maximal current amplitude increased from 1-wk (232 pA) to 9-wk (1025 pA) postdifferentiation. Whole cell I(Ba)s were partially blocked by specific channel blockers to a similar extent in 1- to 3-wk and 7- to 9-wk postdifferentiation NT2-N cells: 10 microM nifedipine (19 vs. 25%), 10 microM conotoxin GVIA (27 vs. 25%), 10 microM conotoxin MVIIC (15 vs. 16%), and 1.75 microM SNX-482 (31 vs. 33%). Currents were completely blocked by 300 microM cadmium. In the presence of nifedipine, GVIA, and MVIIC, approximately 35% of current remained, which was reduced further by SNX-482 (7-14% of current remained), consistent with functional expression of L-, N-, and P/Q-calcium channel types and one or more R-type channel. The presence of multiple calcium currents in this human neuronal-type cell line provides a potentially useful model for study of the regulation, expression and cellular function of human derived calcium channel currents; in particular the R-type current(s).

Vascular smooth muscle cells express the alpha(1A) subunit of a P-/Q-type voltage-dependent Ca(2+)Channel, and It is functionally important in renal afferent arterioles.

In the present study, we tested whether the alpha(1A) subunit, which encodes a neuronal isoform of voltage-dependent Ca(2+) channels (VDCCs) (P-/Q-type), was present and functional in vascular smooth muscle and renal resistance vessels. By reverse transcription-polymerase chain reaction and Southern blotting analysis, mRNA encoding the alpha(1A) subunit was detected in microdissected rat preglomerular vessels and vasa recta, in cultures of rat preglomerular vascular smooth muscle cells (VSMCs), and in cultured rat mesangial cells. With immunoblots, alpha(1A) subunit protein was demonstrated in rat aorta, brain, aortic smooth muscle cells (A7r5), VSMCs, and mesangial cells. Immunolabeling with an anti-alpha(1A) antibody was positive in acid-macerated, microdissected preglomerular vessels and in A7r5 cells. Patch-clamp experiments on aortic A7r5 cells showed 22+/-4% (n=6) inhibition of inward Ca(2+) current by omega-Agatoxin IVA (10(-8) mol/L), which in this concentration is a specific inhibitor of P-type VDCCs. Measurements of intracellular Ca(2+) in afferent arterioles with fluorescence-imaging microscopy showed 32+/-9% (n=10) inhibition of the K(+)-induced rise in Ca(2+) in the presence of 10(-8) mol/L omega-Agatoxin IVA. In microperfused rabbit afferent arterioles, omega-Agatoxin IVA inhibited depolarization-mediated contraction with an EC(50) of 10(-17) mol/L and complete blockade at 10(-14) mol/L. We conclude that the alpha(1A) subunit is expressed in VSMCs from renal preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCCs contribute to Ca(2+) influx in aortic and renal VSMCs and are involved in depolarization-mediated contraction in renal afferent arterioles.