RESUMO
Neuronal voltage-gated N-type (Cav2.2) calcium channels are expressed throughout the nervous system and regulate neurotransmitter release and hence synaptic transmission. They are predominantly modulated via G protein-coupled receptor activated pathways, and the well characterized Gßγ subunits inhibit Cav2.2 currents. Analgesic α-conotoxin Vc1.1, a peptide from predatory marine cone snail venom, inhibits Cav2.2 channels by activating pertussis toxin-sensitive Gi/o proteins via the GABAB receptor (GABA(B)R) and potently suppresses pain in rat models. Using a heterologous GABA(B)R expression system, electrophysiology, and mutagenesis, we showed α-conotoxin Vc1.1 modulates Cav2.2 via a different pathway from that of the GABA(B)R agonists GABA and baclofen. In contrast to GABA and baclofen, Vc1.1 changes Cav2.2 channel kinetics by increasing the rate of activation and shifting its half-maximum inactivation to a more hyperpolarized potential. We then systematically truncated the GABA(B)(1a) C terminus and discovered that removing the proximal carboxyl terminus of the GABA(B)(1a) subunit significantly reduced Vc1.1 inhibition of Cav2.2 currents. We propose a novel mechanism by which Vc1.1 activates GABA(B)R and requires the GABA(B)(1a) proximal carboxyl terminus domain to inhibit Cav2.2 channels. These findings provide important insights into how GABA(B)Rs mediate Cav2.2 channel inhibition and alter nociceptive transmission.
Assuntos
Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio Tipo N/metabolismo , Conotoxinas/metabolismo , Receptores de GABA-B/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/genética , Conotoxinas/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Mutação Puntual/fisiologia , RatosRESUMO
α-Conotoxins Vc1.1 and RgIA are small peptides isolated from the venom of marine cone snails. They have effective anti-nociceptive actions in rat models of neuropathic pain. Pharmacological studies in rodent dorsal root ganglion (DRG) show their analgesic effect is mediated by inhibition of N-type (Ca(v)2.2) calcium channels via a pathway involving γ-aminobutyric acid type B (GABA(B)) receptor. However, there is no direct demonstration that functional GABA(B) receptors are needed for inhibition of the Ca(v)2.2 channel by analgesic α-conotoxins. This study examined the effect of the GABA(B) agonist baclofen and α-conotoxins Vc1.1 and RgIA on calcium channel currents after transient knockdown of the GABA(B) receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNA) targeting GABA(B) subunits R1 and R2. Efficient knockdown of GABA(B) receptor expression at mRNA and protein levels was confirmed by quantitative real time PCR (qRT-PCR) and immunocytochemical analysis, respectively. Whole-cell patch clamp recordings conducted 2-4 days after transfection showed that inhibition of N-type calcium channels in response to baclofen, Vc1.1 and RgIA was significantly reduced in GABA(B) receptor knockdown DRG neurons. In contrast, neurons transfected with a scrambled nontargeting siRNA were indistinguishable from untransfected neurons. In the HEK 293 cell heterologous expression system, Vc1.1 and RgIA inhibition of Ca(v)2.2 channels needed functional expression of both human GABA(B) receptor subunits. Together, these results confirm that GABA(B) receptors must be activated for the modulation of N-type (Ca(v)2.2) calcium channels by analgesic α-conotoxins Vc1.1 and RgIA.
Assuntos
Canais de Cálcio Tipo N/fisiologia , Conotoxinas/farmacologia , Neurônios/efeitos dos fármacos , Receptores de GABA-B/metabolismo , Animais , Baclofeno/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Agonistas dos Receptores de GABA-B/farmacologia , Gânglios Espinais/citologia , Células HEK293 , Humanos , Immunoblotting , Potenciais da Membrana/efeitos dos fármacos , Microscopia Confocal , Neurônios/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , Ratos , Ratos Wistar , Receptores de GABA-B/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Over recent years, the role of matrix vesicles in the initial stages of arterial calcification has been recognized. Matrix calcifying vesicles have been isolated from atherosclerotic arteries and the biochemical composition of calcified vesicles has been studied. No studies have yet been carried out to examine the fine structure of matrix vesicles in order to visualize the features of the consequent stages of their calcification in arteries. In the present work, a high resolution ultrastructural analysis has been employed and the study revealed that matrix vesicles in human atherosclerotic lesions are heterogeneous with two main types which we classified. Type I calcified vesicles were presented by vesicles surrounded by two electron-dense layers and these vesicles were found to be resistant to the calcification process in atherosclerotic lesions in situ. Type II matrix vesicles were presented by vesicles surrounded by several electron-dense layers and these vesicles were found to represent calcifying vesicles in atherosclerotic lesions. To test the hypothesis that calcification of matrix vesicles surrounded by multilayer sheets may occur simply as a physicochemical process, independently from the cell regulation, we produced multilamellar liposomes and induced their calcification in vitro in a manner similar to that occurring in matrix vesicles in atherosclerotic lesions in situ.