Human neuronal stargazin-like proteins, gamma(2), gamma(3) and gamma(4); an investigation of their specific localization in human brain and their influence on Ca(V)2.1 voltage-dependent calcium channels expressed in Xenopus oocytes

Background: Stargazin (gamma(2)) and the closely related gamma(3), and gamma(4) transmembrane proteins are part of a family of proteins that may act as both neuronal voltage-dependent calcium channel (VDCC) gamma subunits and transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproponinc (AMPA) receptor regulatory proteins (TARPs). In this investigation, we examined the distribution patterns of the stargazin-like proteins gamma(2), gamma(3), and gamma(4) in the human central nervous system (CNS). In addition, we investigated whether human gamma(2) or gamma(4) could modulate the electrophysiological properties of a neuronal VDCC complex transiently expressed in Xenopus oocytes.Results: The mRNA encoding human gamma(2) is highly expressed in cerebellum, cerebral cortex, hippocampus and thalamus, whereas gamma(3) is abundant in cerebral cortex and amygdala and gamma(4) in the basal ganglia. Immunohistochemical analysis of the cerebellum determined that both gamma(2) and gamma(4) are present in the molecular layer, particularly in Purkinje cell bodies and dendrites, but have an inverse expression pattern to one another in the dentate cerebellar nucleus. They are also detected in the interneurons of the granule cell layer though only gamma(2) is clearly detected in granule cells. The hippocampus stains for gamma(2) and gamma(4) throughout the layers of the every CA region and the dentate gyrus, whilst gamma(3) appears to be localized particularly to the pyramidal and granule cell bodies. When co-expressed in Xenopus oocytes with a Ca(V)2.1/beta(4) VDCC complex, either in the absence or presence of an alpha(2)delta(2) subunit, neither gamma(2) nor gamma(4) significantly modulated the VDCC peak current amplitude, voltage-dependence of activation or voltage-dependence of steady-state inactivation.Conclusion: The human gamma(2), gamma(3) and gamma(4) stargazin-like proteins are detected only in the CNS and display differential distributions among brain regions and several cell types in found in the cerebellum and hippocampus. These distribution patterns closely resemble those reported by other laboratories for the rodent orthologues of each protein. Whilst the fact that neither gamma(2) nor gamma(4) modulated the properties of a VDCC complex with which they could associate in vivo in Purkinje cells adds weight to the hypothesis that the principal role of these proteins is not as auxiliary subunits of VDCCs, it does not exclude the possibility that they play another role in VDCC function

Voltage-gated calcium channels and their auxiliary subunits: physiology and pathophysiology and pharmacology

Voltage-gated calcium channels are essential players in many physiological processes in excitable cells. There are three main subdivisions of calcium channel, defined by the pore-forming α1 subunit, the CaV 1, CaV 2 and CaV 3 channels. For all the subtypes of voltage-gated calcium channel, their gating properties are key for the precise control of neurotransmitter release, muscle contraction and cell excitability, among many other processes. For the CaV 1 and CaV 2 channels, their ability to reach their required destinations in the cell membrane, their activation and the fine tuning of their biophysical properties are all dramatically influenced by the auxiliary subunits that associate with them. Furthermore, there are many diseases, both genetic and acquired, involving voltage-gated calcium channels. This review will provide a general introduction and then concentrate particularly on the role of auxiliary α2 δ subunits in both physiological and pathological processes involving calcium channels, and as a therapeutic target

How postdoctoral research in Paul Greengard's laboratory shaped my scientific career, although I never did another phosphorylation assay.

In this short review, I describe from personal experience how every step in the career of any scientist, no matter how disjointed and pragmatic each might seem at the time, will almost inevitably meld together, to help us all tackle novel projects. My postdoctoral research in Paul Greengard's lab, where I investigated neurotransmitter-mediated phosphorylation of Synapsin I, was instrumental in my career progression, and Paul's support was instrumental in my ability to make a leap into independent research

The Upregulation of α2δ-1 Subunit Modulates Activity-Dependent Ca2+ Signals in Sensory Neurons.

As auxiliary subunits of voltage-gated Ca(2+) channels, the α2δ proteins modulate membrane trafficking of the channels and their localization to specific presynaptic sites. Following nerve injury, upregulation of the α2δ-1 subunit in sensory dorsal root ganglion neurons contributes to the generation of chronic pain states; however, very little is known about the underlying molecular mechanisms. Here we show that the increased expression of α2δ-1 in rat sensory neurons leads to prolonged Ca(2+) responses evoked by membrane depolarization. This mechanism is coupled to CaV2.2 channel-mediated responses, as it is blocked by a ω-conotoxin GVIA application. Once initiated, the prolonged Ca(2+) transients are not dependent on extracellular Ca(2+) and do not require Ca(2+) release from the endoplasmic reticulum. The selective inhibition of mitochondrial Ca(2+) uptake demonstrates that α2δ-1-mediated prolonged Ca(2+) signals are buffered by mitochondria, preferentially activated by Ca(2+) influx through CaV2.2 channels. Thus, by controlling channel abundance at the plasma membrane, the α2δ-1 subunit has a major impact on the organization of depolarization-induced intracellular Ca(2+) signaling in dorsal root ganglion neurons

Alternative Splicing in CaV2.2 Regulates Neuronal Trafficking via Adaptor Protein Complex-1 Adaptor Protein Motifs

UNLABELLED: N-type voltage-gated calcium (CaV2.2) channels are expressed in neurons and targeted to the plasma membrane of presynaptic terminals, facilitating neurotransmitter release. Here, we find that the adaptor protein complex-1 (AP-1) mediates trafficking of CaV2.2 from the trans-Golgi network to the cell surface. Examination of splice variants of CaV2.2, containing either exon 37a (selectively expressed in nociceptors) or 37b in the proximal C terminus, reveal that canonical AP-1 binding motifs, YxxΦ and [DE]xxxL[LI], present only in exon 37a, enhance intracellular trafficking of exon 37a-containing CaV2.2 to the axons and plasma membrane of rat DRG neurons. Finally, we identify differential effects of dopamine-2 receptor (D2R) and its agonist-induced activation on trafficking of CaV2.2 isoforms. D2R slowed the endocytosis of CaV2.2 containing exon 37b, but not exon 37a, and activation by the agonist quinpirole reversed the effect of the D2R. Our work thus reveals key mechanisms involved in the trafficking of N-type calcium channels. SIGNIFICANCE STATEMENT: CaV2.2 channels are important for neurotransmitter release, but how they are trafficked is still poorly understood. Here, we describe a novel mechanism for trafficking of CaV2.2 from the trans-Golgi network to the cell surface which is mediated by the adaptor protein AP-1. Alternative splicing of exon 37 produces CaV2.2-exon 37a, selectively expressed in nociceptors, or CaV2.2-exon 37b, which is the major splice isoform. Our study reveals that canonical AP-1 binding motifs (YxxΦ and [DE]xxxL[LI]), present in exon 37a, but not 37b, enhance intracellular trafficking of exon 37a-containing CaV2.2 to axons and plasma membrane of DRG neurons. Interaction of APs with CaV2.2 channels may also be key underlying mechanisms for differential effects of the dopamine D2 receptor on trafficking of CaV2.2 splice variants

Rab11-dependent recycling of calcium channels is mediated by auxiliary subunit α2δ-1 but not α2δ-3

N-type voltage-gated calcium channels (CaV2.2) are predominantly expressed at presynaptic terminals, and their function is regulated by auxiliary α2δ and β subunits. All four mammalian α2δ subunits enhance calcium currents through CaV1 and CaV2 channels, and this increase is attributed, in part, to increased CaV expression at the plasma membrane. In the present study we provide evidence that α2δ-1, like α2δ-2, is recycled to the plasma membrane through a Rab11a-dependent endosomal recycling pathway. Using a dominant-negative Rab11a mutant, Rab11a(S25N), we show that α2δ-1 increases plasma membrane CaV2.2 expression by increasing the rate and extent of net forward CaV2.2 trafficking in a Rab11a-dependent manner. Dominant-negative Rab11a also reduces the ability of α2δ-1 to increase CaV2.2 expression on the cell-surface of hippocampal neurites. In contrast, α2δ-3 does not enhance rapid forward CaV2.2 trafficking, regardless of whether Rab11a(S25N) is present. In addition, whole-cell CaV2.2 currents are reduced by co-expression of Rab11a(S25N) in the presence of α2δ-1, but not α2δ-3. Taken together these data suggest that α2δ subtypes participate in distinct trafficking pathways which in turn influence the localisation and function of CaV2.2

Fight or flight: The culprit is lurking in the neighbourhood

The fight-or-flight response is studied by all students of Physiology as a concerted bodily response to danger. Liu et al (2020) have now revealed its mechanism, after surveying the proteomic neighbourhood around the cardiac calcium channels in a study which is a tour-de-force of modern biological techniques

The CaVβ Subunit Protects the I-II Loop of the Voltage-gated Calcium Channel CaV2.2 from Proteasomal Degradation but Not Oligoubiquitination.

CaVβ subunits interact with the voltage-gated calcium channel CaV2.2 on a site in the intracellular loop between domains I and II (the I-II loop). This interaction influences the biophysical properties of the channel and leads to an increase in its trafficking to the plasma membrane. We have shown previously that a mutant CaV2.2 channel that is unable to bind CaVβ subunits (CaV2.2 W391A) was rapidly degraded (Waithe, D., Ferron, L., Page, K. M., Chaggar, K., and Dolphin, A. C. (2011) J. Biol. Chem. 286, 9598-9611). Here we show that, in the absence of CaVβ subunits, a construct consisting of the I-II loop of CaV2.2 was directly ubiquitinated and degraded by the proteasome system. Ubiquitination could be prevented by mutation of all 12 lysine residues in the I-II loop to arginines. Including a palmitoylation motif at the N terminus of CaV2.2 I-II loop was insufficient to target it to the plasma membrane in the absence of CaVβ subunits even when proteasomal degradation was inhibited with MG132 or ubiquitination was prevented by the lysine-to-arginine mutations. In the presence of CaVβ subunit, the palmitoylated CaV2.2 I-II loop was protected from degradation, although oligoubiquitination could still occur, and was efficiently trafficked to the plasma membrane. We propose that targeting to the plasma membrane requires a conformational change in the I-II loop that is induced by binding of the CaVβ subunit

The α2δ subunits of voltage-gated calcium channels.

Voltage-gated calcium channels consist of the main pore-forming α1 subunit, together, except in the case of the T-type channels, with β and α2δ and sometimes γ subunits, which are collectively termed auxiliary or accessory subunits. This review will concentrate on the properties and role of the α2δ subunits of these channels. These proteins are largely extracellular, membrane-associated proteins which influence the trafficking, localization, and biophysical properties of the channels. This article is part of a Special Issue entitled: Calcium channels

Voltage-gated calcium channels: their discovery, function and importance as drug targets

This review will first describe the importance of Ca2+ entry for function of excitable cells, and the subsequent discovery of voltage-activated calcium conductances in these cells. This finding was rapidly followed by the identification of multiple subtypes of calcium conductance in different tissues. These were initially termed low- and high-voltage activated currents, but were then further subdivided into L-, N-, PQ-, R and T-type calcium currents on the basis of differing pharmacology, voltage-dependent and kinetic properties, and single channel conductance. Purification of skeletal muscle calcium channels allowed the molecular identification of the pore-forming and auxiliary α2δ, β and ϒ subunits present in these calcium channel complexes. These advances then led to the cloning of the different subunits, which permitted molecular characterisation, to match the cloned channels with physiological function. Studies with knockout and other mutant mice then allowed further investigation of physiological and pathophysiological roles of calcium channels. In terms of pharmacology, cardiovascular L-type channels are targets for the widely used antihypertensive 1,4-dihydropyridines and other calcium channel blockers, N-type channels are a drug target in pain, and α2δ-1 is the therapeutic target of the gabapentinoid drugs, used in neuropathic pain. Recent structural advances have allowed a deeper understanding of Ca2+ permeation through the channel pore and the structure of both the pore-forming and auxiliary subunits. Voltage-gated calcium channels are subject to multiple pathways of modulation by G-protein and second messenger regulation. Furthermore their trafficking pathways, subcellular localisation and functional specificity are the subjects of active investigation