Biology subjects

Dolphin, A. C.

Publications and source records attributed to Dolphin, A. C..

7 recordsLinked to original sources

bioRxiv2023

The interplay between splicing of exons 18a and 47 differentially affects membrane targeting and function of human CaV2.2

N-type calcium channels (CaV2.2) are predominantly localized in presynaptic terminals, and are particularly important for pain transmission in the spinal cord. Furthermore, they have multiple isoforms, conferred by alternatively-spliced or cassette exons, which are differentially expressed. Here we have examined alternatively-spliced exon47 variants that encode a long or short C-terminus in human CaV2.2. In the Ensembl database, all short exon47-containing transcripts were associated with the absence of exon 18a, therefore we also examined effect of inclusion or absence of exon18a, combinatorially with the exon47 splice variants. We found that long exon47, only in the additional presence of exon18a, results in CaV2.2 currents that have a 3.6-fold greater maximum conductance than the other three combinations. In contrast, cell surface expression of CaV2.2 in both tsA-201 cells and hippocampal neurons is increased [~]4-fold by long exon47 relative to short exon47, in either the presence or absence of exon18a. This surprising discrepancy between trafficking and function indicates that cell surface expression is enhanced by long exon47, independently of exon 18a. However, in the presence of exon47, exon18a mediates an additional permissive effect on CaV2.2 gating. We also investigated the SNP in exon47 that has been linked to schizophrenia and Parkinsons disease, which we found is only non-synonymous in the short exon47 C-terminal isoform, resulting in two minor alleles. This study highlights the importance of investigating the combinatorial effects of exon inclusion, rather than each in isolation, in order to increase our understanding of calcium channel function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/557884v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@132c8fborg.highwire.dtl.DTLVardef@12910ccorg.highwire.dtl.DTLVardef@10de072org.highwire.dtl.DTLVardef@a86f7_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Capsaicin-induced endocytosis of endogenous presynaptic CaV2.2 in DRG-spinal cord co-cultures inhibits presynaptic function

The N-type calcium channel, CaV2.2 is key to neurotransmission from the primary afferent terminals of dorsal root ganglion (DRG) neurons to their post-synaptic targets in the spinal cord. In this study we have utilized CaV2.2_HA knock-in mice, because the exofacial epitope tag in CaV2.2_HA enables accurate detection and localization of endogenous CaV2.2. CaV2.2_HA knock-in mice were used as a source of DRGs to exclusively study the presynaptic expression of N-type calcium channels in co-cultures between DRG neurons and wild-type spinal cord neurons. CaV2.2_HA is strongly expressed on the cell surface, particularly in TRPV1-positive small and medium DRG neurons. Super-resolution images of the presynaptic terminals revealed an increase in CaV2.2_HA expression and increased association with the post-synaptic marker Homer over time in vitro. Brief application of the TRPV1 agonist, capsaicin, resulted in a significant down-regulation of cell surface CaV2.2_HA expression in DRG neuron somata. At their presynaptic terminals, capsaicin caused a reduction in CaV2.2_HA proximity to and co-localization with the active zone marker RIM 1/2, as well as a lower contribution of N-type channels to single action potential-mediated Ca2+ influx. The mechanism of this down-regulation of CaV2.2_HA involves a Rab 11a-dependent trafficking process, since dominant-negative Rab11a(S25N) occludes the effect of capsaicin on presynaptic CaV2.2_HA expression, and also prevents the effect of capsaicin on action potential induced Ca2+ influx. Taken together, these data suggest that capsaicin causes a decrease in cell surface CaV2.2_HA expression in DRG terminals via a Rab11a-dependent endosomal trafficking pathway.

neuroscience↗

bioRxiv2022

Involvement of CaV2.2 channels and α2δ-1 in hippocampal homeostatic synaptic plasticity

In the mammalian brain, presynaptic CaV2 channels play a pivotal role for synaptic transmission by mediating fast neurotransmitter exocytosis via influx of Ca2+ into the active zone of presynaptic terminals. However, the distribution and modulation of CaV2.2 channels at highly plastic hippocampal synapses remains to be elucidated. Here, we assess CaV2.2 channels during homeostatic synaptic plasticity, a compensatory form of homeostatic control preventing excessive or insufficient neuronal activity during which extensive active zone remodelling has been described. We show that chronic silencing of neuronal activity in mature hippocampal cultures resulted in elevated presynaptic Ca2+ transients, mediated by increased levels of CaV2.2 channels at the presynaptic site. This work focussed further on the role of 2{delta}-1 subunits, important regulators of synaptic transmission and CaV2.2 channel abundance at the presynaptic membrane. We find that 2{delta}-1-overexpression reduces the contribution of CaV2.2 channels to total Ca2+ flux without altering the amplitude of the Ca2+ transients. Levels of endogenous 2{delta}-1 decreased during homeostatic synaptic plasticity, whereas the overexpression of 2{delta}-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV2.2 channels and novel roles for 2{delta}-1 during synaptic plastic adaptation.

neuroscience↗

bioRxiv2021

T-type Ca2+ and persistent Na+ currents synergistically elevate ventral, not dorsal, entorhinal cortical stellate cell excitability

The medial entorhinal cortex (mEC) plays a salient role in physiological processes such as spatial cognition and spatial coding. mEC layer II stellate neurons, in particular, influence these processes. Interestingly, ventral and dorsal stellate neurons diversely affect these processes and have distinct intrinsic membrane properties and action potential firing patterns. Little, though, is known about how ventral stellate neuron intrinsic excitability is regulated. We show that ventral stellate neurons predominantly possess T-type Ca2+ currents encoded by CaV3.2 subunits, with dorsal stellate neurons having small or no currents. Further, twice as much CaV3.2 mRNA was present in ventral than dorsal mEC. In line with T-type, CaV3.2 Ca2+ current biophysical properties, depolarising stimuli activated these currents in ventral, but not dorsal, neurons. Here, these currents acted in concert with persistent Na+ currents to elevate input resistance and tonic action potential firing. CaV3.2 currents also enhanced excitatory post-synaptic potential decay and integration solely in ventral neurons. These results reveal that CaV3.2 currents, together with persistent Na+ currents, impart the characteristic intrinsic membrane and firing properties of ventral stellate neurons. This signifies that specific voltage-gated conductances distinctly affect ventral and dorsal mEC stellate neuron activity and functions such as spatial memory and spatial navigation.

neuroscience↗

bioRxiv2021

Amino acid sensor conserved from bacteria to humans

Amino acids are recognized as signals by various receptors in bacteria, archaea, and eukaryotes. However, no common mechanism for amino acid recognition is currently known. Here we show that a subclass of a ubiquitous extracellular domain dCache_1 contains a simple amino acid recognition motif, and it is found throughout the Tree of Life. In bacteria, this motif exclusively binds amino acids, including GABA, and it is present in all major receptor types. In humans, this motif is found in 2{delta} subunits of voltage-gated calcium channels that are implicated in neuropathic pain and neurodevelopmental disorders. Our findings suggest that GABA-derived drugs bind to the same motif in human 2{delta} subunits that binds natural GABA ligands in bacterial chemoreceptors.

evolutionary biology↗

bioRxiv2021

Identification that ADAM17 mediates proteolytic maturation of calcium channel auxiliary α2δ subunits, and enables calcium current enhancement

The auxiliary 2{delta} subunits of voltage-gated calcium (CaV) channels are key to augmenting expression and function of CaV1 and CaV2 channels, and are also important drug targets in several therapeutic areas, including neuropathic pain. The 2{delta} proteins are translated as pre-proteins encoding both 2 and {delta}, and post-translationally proteolysed into 2 and {delta} subunits, which remain associated as a complex. In this study we have identified ADAM17 as a key protease involved in proteolytic processing of pro-2{delta}-1 and 2{delta}-3 subunits. We provide three lines of evidence: firstly, proteolytic cleavage is inhibited by chemical inhibitors of particular metalloproteases, including ADAM17. Secondly, proteolytic cleavage of both 2{delta}-1 and 2{delta}-3 is markedly reduced in cell lines by knockout of ADAM17 but not ADAM10. Thirdly, proteolytic cleavage is reduced by the N-terminal active domain of TIMP-3 (N-TIMP-3), which selectively inhibits ADAM17. We have found previously that proteolytic cleavage into mature 2{delta} is essential for the enhancement of CaV function, and in agreement, knockout of ADAM17 inhibited the ability of 2{delta}-1 to enhance both CaV2.2 and CaV1.2 calcium currents. Thus, our study identifies ADAM17 as a key protease required for proteolytic maturation of 2{delta}-1 and 2{delta}-3, and thus a potential drug target in neuropathic pain.

neuroscience↗

bioRxiv2021

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 2{delta} and {beta} subunits. All four mammalian 2{delta} 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 2{delta}-1, like 2{delta}-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 2{delta}-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 2{delta}-1 to increase CaV2.2 expression on the cell-surface of hippocampal neurites. In contrast, 2{delta}-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 2{delta}-1, but not 2{delta}-3. Taken together these data suggest that 2{delta} subtypes participate in distinct trafficking pathways which in turn influence the localisation and function of CaV2.2. Summary statementThe calcium channel auxiliary subunit 2{delta}-1 but not 2{delta}-3 participates in Rab11a-dependent recycling, which in turn influences the localisation and function of CaV2.2.

neuroscience↗