Abstract:Background: We examined the role of membrane anchoring of voltage-gated calcium channel α2δ subunits.Results: We used a truncated α2δ-1 construct (α2δ-1ΔC-term), which still increases CaV2.1/β1b currents, despite being mainly secreted.Conclusion: The effect of α2δ-1ΔC-term on calcium currents does not involve secretion and subsequent re-binding to the plasma membrane.Significance: C-terminal membrane anchoring of α2δ is not essential for calcium current enhancement.
“…We therefore examined the cell-surface expression of α 2 δ-1-HA, and the effect of Ca V 2.2 coexpression on this, to gain insight into the interaction site of the auxiliary subunits, with the aim of determining whether they remained associated as judged by colocalization at the cell surface. We used α 2 δ-1-HA for these studies, as we have shown previously that it supports calcium channel currents with identical properties to WT α 2 δ-1 (15). Surprisingly, we found that the amount of α 2 δ-1-HA detected on the cell surface was strongly reduced by coexpression of Ca V 2.2-BBS, both in the presence and absence of the β subunit ( Fig.…”
Ca V 1 and Ca V 2 voltage-gated calcium channels are associated with β and α 2 δ accessory subunits. However, examination of cell surface-associated Ca V 2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged Ca V 2 channels. Here we report the development of fully functional Ca V 2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neuronal cell line and neurons. We first examined the effect of the auxiliary subunits. Although α 2 δ subunits copurify with Ca V 2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether α 2 δ subunits are associated with these channels at the cell surface. We found that, whereas α 2 δ-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with Ca V 2.2/β1b, despite our finding that α 2 δ-1 increases plasmamembrane Ca V 2.2 expression. However, this was due to occlusion of the antigenic epitope by association with Ca V 2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between α 2 δ-1 and the α 1 subunit at the plasma membrane. We further show that, although Ca V 2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type α 2 δ-1 (but not a gabapentin-insensitive α 2 δ-1 mutant), the interaction between Ca V 2.2 and α 2 δ-1 is not disrupted by gabapentin. Altogether, these results demonstrate that Ca V 2.2 and α 2 δ-1 are intimately associated at the plasma membrane and allow us to infer a region of interaction. P urification of L-type voltage-gated calcium (Ca V ) channels from skeletal muscle shows that they consist of a pore-forming α 1 subunit, Ca V 1.1, associated with three accessory subunits, β1, α 2 δ-1, and γ 1 (1, 2). Cardiac L-type channels have a similar subunit composition, although the α 1 subunit is α1C and the γ subunit is not present (3). However, the study of cell surface-associated N-type (Ca V 2.2) and P/Q-type (Ca V 2.1) calcium channels has been hampered by the lack both of antibodies to cell-surface epitopes and of functional exofacially tagged Ca V 2 channels. Here we report the development of fully functional Ca V 2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the cell surface, in both cell lines and neurons. Using this methodological advance, we can now examine directly the effect of the auxiliary subunits on cellsurface expression of Ca V 2 channels.Although α 2 δ subunits have been shown to be associated with Ca V 2.1 and Ca V 2.2 following purification (4, 5), it has recently been suggested that the α 2 δ subunits are associated only very loosely and nonquantitatively with Ca V 2 channels (6), calling into question their role as calcium channel subunits. This study found that the α 2 δ proteins α 2 δ-1, α 2 δ-2, and α 2 δ-3 c...
“…We therefore examined the cell-surface expression of α 2 δ-1-HA, and the effect of Ca V 2.2 coexpression on this, to gain insight into the interaction site of the auxiliary subunits, with the aim of determining whether they remained associated as judged by colocalization at the cell surface. We used α 2 δ-1-HA for these studies, as we have shown previously that it supports calcium channel currents with identical properties to WT α 2 δ-1 (15). Surprisingly, we found that the amount of α 2 δ-1-HA detected on the cell surface was strongly reduced by coexpression of Ca V 2.2-BBS, both in the presence and absence of the β subunit ( Fig.…”
Ca V 1 and Ca V 2 voltage-gated calcium channels are associated with β and α 2 δ accessory subunits. However, examination of cell surface-associated Ca V 2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged Ca V 2 channels. Here we report the development of fully functional Ca V 2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neuronal cell line and neurons. We first examined the effect of the auxiliary subunits. Although α 2 δ subunits copurify with Ca V 2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether α 2 δ subunits are associated with these channels at the cell surface. We found that, whereas α 2 δ-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with Ca V 2.2/β1b, despite our finding that α 2 δ-1 increases plasmamembrane Ca V 2.2 expression. However, this was due to occlusion of the antigenic epitope by association with Ca V 2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between α 2 δ-1 and the α 1 subunit at the plasma membrane. We further show that, although Ca V 2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type α 2 δ-1 (but not a gabapentin-insensitive α 2 δ-1 mutant), the interaction between Ca V 2.2 and α 2 δ-1 is not disrupted by gabapentin. Altogether, these results demonstrate that Ca V 2.2 and α 2 δ-1 are intimately associated at the plasma membrane and allow us to infer a region of interaction. P urification of L-type voltage-gated calcium (Ca V ) channels from skeletal muscle shows that they consist of a pore-forming α 1 subunit, Ca V 1.1, associated with three accessory subunits, β1, α 2 δ-1, and γ 1 (1, 2). Cardiac L-type channels have a similar subunit composition, although the α 1 subunit is α1C and the γ subunit is not present (3). However, the study of cell surface-associated N-type (Ca V 2.2) and P/Q-type (Ca V 2.1) calcium channels has been hampered by the lack both of antibodies to cell-surface epitopes and of functional exofacially tagged Ca V 2 channels. Here we report the development of fully functional Ca V 2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the cell surface, in both cell lines and neurons. Using this methodological advance, we can now examine directly the effect of the auxiliary subunits on cellsurface expression of Ca V 2 channels.Although α 2 δ subunits have been shown to be associated with Ca V 2.1 and Ca V 2.2 following purification (4, 5), it has recently been suggested that the α 2 δ subunits are associated only very loosely and nonquantitatively with Ca V 2 channels (6), calling into question their role as calcium channel subunits. This study found that the α 2 δ proteins α 2 δ-1, α 2 δ-2, and α 2 δ-3 c...
“…This finding implies that all α 2 δ-1 is proteolytically processed prior to intracellular trafficking into DRG axons. Indeed, our biochemical and cell fractionation data indicate that proteolytic cleavage occurs mainly during trafficking to the plasma membrane (Kadurin et al, 2012) (data not shown). However, this result opens the possibility that uncleaved α 2 δ-1 may reach the cell surface in DRGs cell bodies and have a functional role there.10.7554/eLife.21143.019Figure 6.Proteolytic processing of endogenous α 2 δ-1 and effect of exogenous expression of α 2 δ-1 and α 2 (3C)δ-1 in DRGs.( a ) DRGs, spinal nerves and dorsal roots from rats, 4 days after SNL, were dissected and segmented according to the diagram.…”
Section: Resultsmentioning
confidence: 90%
“…We have previously shown that the amount of proteolytically-processed α 2 δ-1 is increased on the cell surface compared to the WCL in transfected tsA-201 cells (Kadurin et al, 2012). However, the present results show that this processing of α 2 δ-1 is not essential for its trafficking to the plasma membrane.…”
The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes.DOI:
http://dx.doi.org/10.7554/eLife.21143.001
“…28 It is not clear however, whether similar increases would be evident in neurons that natively express calcium channels with auxiliary subunits. Surprisingly, 29 expression of neither b4 alone nor a 2 d-1 alone increased current density in SCG neurons (Fig. 8).…”
Type two voltage gated calcium (Ca V 2) channels are the primary mediators of neurotransmission at neuronal presynapses, but their function at neural soma is also important in regulating excitability. 1 Mechanisms that regulate Ca V 2 channel expression at synapses have been studied extensively, which motivated us to perform similar studies in the soma. Rat sympathetic neurons from the superior cervical ganglion (SCG) natively express Ca V 2.2 and Ca V 2.3. 2 We noted previously that heterologous expression of Ca V 2.1 but not Ca V 2.2 results in increased calcium current in SCG neurons. 3 In the present study, we extended these observations to show that both Ca V 2.1 and Ca V 2.3 expression resulted in increased calcium currents while Ca V 2.2 expression did not. Further, Ca V 2.1 could displace native Ca V 2.2 channels, but Ca V 2.3 expression could not. Heterologous expression of the individual accessory subunits a 2 d-1, a 2 d-2, a 2 d-3, or b4 alone failed to increase current density, suggesting that the calcium current ceiling when Ca V 2.2 was over-expressed was not due to lack of these subunits. Interestingly, introduction of recombinant a2d subunits produced surprising effects on displacement of native Ca V 2.2 by recombinant channels. Both a 2 d-1 and a 2 d-2 seemed to promote Ca V 2.2 displacement by recombinant channel expression, while a 2 d-3 appeared to protect Ca V 2.2 from displacement. Thus, we observe a selective prioritization of Ca V channel functional expression in neurons by specific a 2 d subunits. These data highlight a new function for a 2 d subtypes that could shed light on subtype selectivity of Ca V 2 membrane expression.
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