Pain Perception in Mice Lacking the β3 Subunit of Voltage-activated Calcium Channels

Abstract

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N-and P/Q-type calcium channels as well as by gene targeting of the ␣1B subunit (N-type). The accessory ␤3 subunits of calcium channels are preferentially associated with the ␣1B subunit in neurones. Here we show that deletion of the ␤3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cord in vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of ␤3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (␣1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.Voltage-activated calcium channels represent a family of ionic channels that play a crucial role in the nervous system by controlling membrane excitability and neurotransmitter release. Neurones express L-, N-, P/Q-, and R-type calcium channels, which are composed of the pore-forming subunit ␣1 and the accessory subunits ␤, ␣2␦, (1, 2) and probably ␥ (3). The importance of ␣1D (L-type), ␣1B (N-type), ␣1A (P/Q-type), and ␣1E (probably R-type) for the function of the nervous system in vivo is underlined by recent studies with genetically engineered mice (4). By contrast, the in vivo function of the four known ␤ subunits (␤1, ␤2, ␤3, ␤4) of calcium channels is less understood. In heterologous expression systems, ␤ subunits are required for the functional expression of ␣1 subunits (5-7), enhance the current density, and shift the voltage dependence of the activation and inactivation of recombinant calcium channels (8 -11). Additionally, ␤ subunits have been implicated in the modulation of calcium channels by G proteins (12-14). In vivo, the expression of ␤ subunits is modified in pathological states such as cardiac dysfunction (15) and diabetes (16). A natural occurring mutation of ␤4 is associated with ataxia and absence seizures in lethargic (lh/lh) mice, and this mutation modifies N-and P/Q-type calcium channels in the brain (17). The targeted disruption of ␤1 is lethal (18). In sympathetic neurones of ␤3-deficient mice, N-and L-type calcium channel currents are reduced, the activation of P/Q type calcium channels is altered, and no difference appears concerning inhibitory effects of norepinephrine on calcium channel currents (19). Such alterations of calcium channel currents in sympathetic neurones might be related to the cardiovascular phenotype of mice lacking the ␤3 subunit (20). Yet, the in vivo functions of the ␤3 subunit of ...

Modified Sympathetic Nerve System Activity with Overexpression of the Voltage-dependent Calcium Channel β3 Subunit

“…The effects of the ␤ subunit on N-type channel populations have been reported in null-mutant mice (16), whereas in vivo overexpression experiments, such as a transgenic approach, have failed. Combined with our previous study (16), the evidence clearly indicates that ␤3 has a strong influence on N-type channel populations. Furthermore, the protein level of CaV2.2 in the microsomal fraction was apparently dependent on the co-expressed ␤3 subunit (supplemental Fig.…”

“…The ␤3-deficient (␤3 Ϫ/Ϫ ) and the CaV2.2-deificient mice were generated by gene-targeting methods (11,16). All experiments were conducted in accordance with the Guidelines for the Use of Laboratory Animals of the Akita University School of Medicine.…”

“…Currents were recorded with an Axopatch 200B patch clamp amplifier (Axon Instruments) (16). Currents were elicited by voltage clamp steps from Ϫ80 mV every 15 s. To examine channel components, -agatoxin IVA (0.1 M), -conotoxin GVIA (1 M), nimodipine (10 M), and nickel (100 M) were used.…”

Identification and expression of voltage‐gated calcium channel β subunits in Zebrafish

“…Thus, The mammalian CACNB3 gene is mainly expressed in the brain but also in a variety of other tissues, including the aorta, trachea, lung, heart, pancreas, and adrenal gland (Hullin et al, 1992;Castellano et al, 1993). CACNB3 knockout mice displayed reduced nociception (Murakami et al, 2002), which was attributed to the reduction of high voltage-gated Ca 2ϩ currents in dorsal root ganglion cells. A similar reduction of voltage-gated Ca 2ϩ currents was also observed in sympathetic neurons in CACNB3 knockout mice (Namkung et al, 1998).…”

Les canaux calciques dépendants du voltage au cœur de la douleur

“…En effet, l'invalidation génique de la sousunité β 3 chez l'animal semble conférer à ces animaux un seuil de sensibilité accru à la douleur inflammatoire [33]. Il est toutefois probable que cette sous-unité ne soit pas impliquée de manière directe dans les méca-nismes de nociception, mais vraisemblablement via les canaux auxquels elle est associée.…”