Spinal morphine but not ziconotide or gabapentin analgesia is affected by alternative splicing of voltage-gated calcium channel CaV2.2 pre-mRNA

Presynaptic voltage-gated calcium CaV2.2 channels play a privileged role in spinal level sensitization following peripheral nerve injury. Direct and indirect inhibitors of CaV2.2 channel activity in spinal dorsal horn are analgesic in chronic pain states. CaV2.2 channels represent a family of splice isoforms that are expressed in different combinations according to cell-type. A pair of mutually exclusive exons in the CaV2.2 encoding Cacna1b gene, e37a and e37b, differentially influence morphine analgesia. In mice that lack exon e37a, which is enriched in nociceptors, the analgesic efficacy of intrathecal morphine against noxious thermal stimuli is reduced. Here we ask if sequences unique to e37a influence: the development of abnormal thermal and mechanical sensitivity associated with peripheral nerve injury; and the actions of two other classes of analgesics that owe part or all of their efficacy to CaV2.2 channel inhibition. We find that: i) the analgesic efficacy of morphine, but not ziconotide or gabapentin, is reduced in mice lacking e37a, ii) the induction and maintenance of behaviors associated with sensitization that accompany peripheral nerve injury, do not require e37a-specific sequence, iii) intrathecal morphine, but not ziconotide or gabapentin analgesia to thermal stimuli is significantly lower in wild-type mice after peripheral nerve injury, iv) the analgesic efficacy of ziconotide and gabapentin to mechanical stimuli is reduced following nerve injury, and iv) intrathecal morphine analgesia to thermal stimuli in mice lacking e37a is not further reduced by peripheral nerve injury. Our findings show that the analgesic action of morphine, but not ziconotide or gabapentin, to thermal stimuli is linked to which Cacna1b exon, e37a or e37b, is selected during alternative pre-mRNA splicing

Identification of Functionally Distinct Isoforms of the N-Type Ca2+ Channel in Rat Sympathetic Ganglia and Brain

AbstractThe N channel is critical for regulating release of neurotransmitter at many synapses, and even subtle differences in its activity would be expected to influence the efficacy of synaptic transmission. Although several splice variants of the N channel are expressed in the mammalian nervous system, their biological importance is presently unclear. Here, we show that variants of the α1B subunit of the N channel are expressed in sympathetic ganglia and that alternative splicing within IIIS3-S4 and IVS3-S4 generate kinetically distinct channels. We further show a striking difference between the expression pattern of the S3-S4 variants in brain and peripheral ganglia and conclude that the brain-dominant form of the N channel gates 2- to 4-fold more rapidly than that predominant in ganglia

Constitutive activity of the Ghrelin receptor reduces surface expression of voltage-gated Ca2+ channels in a CaVβ-dependent manner

Voltage-gated Ca2+ (CaV) channels couple membrane depolarization to Ca2+ influx, triggering a range ofCa2+-dependent cellular processes. CaV channels are, therefore, crucial in shaping neuronal activity and function, depending on their individual temporal and spatial properties. Furthermore, many neurotransmitters and drugs that act through G protein coupled receptors (GPCRs), modulate neuronal activity by altering the expression, trafficking, or function of CaV channels. GPCRdependent mechanisms that downregulate CaV channel expression levels are observed in many neurons but are, by comparison, less studied. Herewe showthat the growth hormone secretagogue receptor type 1a (GHSR), a GPCR, can inhibit the forwarding trafficking of severalCaV subtypes, even in the absence of agonist. This constitutive form ofGPCRinhibition of CaV channels depends on the presence of a CaVβ subunit. CaVβ subunits displace CaVα1 subunits from the endoplasmic reticulum. The actions of GHSR on CaV channels trafficking suggest a role for this signaling pathway in brain areas that control food intake, reward, and learning and memory.Instituto Multidisciplinario de Biología Celula

Cellular/Molecular Differential Ubiquitination and Proteasome Regulation of Ca V 2.2 N-Type Channel Splice Isoforms

Ca V 2.2 (N-type) calcium channels control the entry of calcium into neurons to regulate essential functions but most notably presynaptic transmitter release. Ca V 2.2 channel expression levels are precisely controlled, but we know little of the cellular mechanisms involved. The ubiquitin proteasome system (UPS) is known to regulate expression of many synaptic proteins, including presynaptic elements, to optimize synaptic efficiency. However, we have limited information about ubiquitination of Ca V 2 channels. Here we show that Ca V 2.2 proteins are ubiquitinated, and that elements in the proximal C terminus of Ca V 2.2 encoded by exon 37b of the mouse Cacna1b gene predispose cloned and native channels to downregulation by the UPS. Ca V 2.2 channels containing e37b are expressed throughout the mammalian nervous system, but in some cells, notably nociceptors, sometimes e37a-not e37b-is selected during alternative splicing of Ca V 2.2 pre-mRNA. By a combination of biochemical and functional analyses we show e37b promotes a form of ubiquitination that is coupled to reduced Ca V 2.2 current density and increased sensitivity to the UPS. Cell-specific alternative splicing of e37a in nociceptors reduces Ca V 2.2 channel ubiquitination and sensitivity to the UPS, suggesting a role in pain processing

Constitutive activity of the Ghrelin receptor reduces surface expression of voltage-gated Ca2+ channels in a CaVβ-dependent manner

Voltage-gated Ca2+ (CaV) channels couple membrane depolarization to Ca2+ influx, triggering a range ofCa2+-dependent cellular processes. CaV channels are, therefore, crucial in shaping neuronal activity and function, depending on their individual temporal and spatial properties. Furthermore, many neurotransmitters and drugs that act through G protein coupled receptors (GPCRs), modulate neuronal activity by altering the expression, trafficking, or function of CaV channels. GPCRdependent mechanisms that downregulate CaV channel expression levels are observed in many neurons but are, by comparison, less studied. Herewe showthat the growth hormone secretagogue receptor type 1a (GHSR), a GPCR, can inhibit the forwarding trafficking of severalCaV subtypes, even in the absence of agonist. This constitutive form ofGPCRinhibition of CaV channels depends on the presence of a CaVβ subunit. CaVβ subunits displace CaVα1 subunits from the endoplasmic reticulum. The actions of GHSR on CaV channels trafficking suggest a role for this signaling pathway in brain areas that control food intake, reward, and learning and memory.Instituto Multidisciplinario de Biología Celula