Functional properties and modulation of extracellular epitope - tagged Ca<sub>V</sub>2.1 voltage-gated calcium channels

Watschinger, Katrin; Horak, Silja B; Schulze, Katrin; Obermair, Gerald J.; Wild, Claudia; Koschak, Alexandra; Sinnegger-Brauns, Martina J.; Tampé, Robert; Striessnig, Jörg

doi:10.4161/chan.2.6.6793

Cited by 26 publications

(40 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The functional exofacially tagged Ca V 2.2 constructs described here thus represent important tools for the examination of Ca V 2.2 distribution and trafficking and the effect of auxiliary subunits and other factors. Expression of Ca V 2.2-HA in DRG neurons also results in robust expression on the cell surface, unlike the finding for an HA-tagged Ca V 2.1 construct (27), providing evidence that these constructs represent important tools for studying Ca V 2.2 trafficking and localization in these neurons.…”

Section: Discussionmentioning

confidence: 79%

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α ₂ δ-1 subunits

Cassidy

Ferron

Kadurin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

131

203

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

confidence: 79%

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α ₂ δ-1 subunits

Cassidy

Ferron

Kadurin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

131

203

View full text Add to dashboard Cite

show abstract

“…The GK domain forms the high-affinity binding pocket for the cytoplasmic ␣ interaction domain in the Ca V 1 and Ca V 2 ␣ 1 subunits Opatowsky et al, 2004;Van Petegem et al, 2004). Alternative splicing of the exons encoding the vari-able regions gives rise to multiple variants of each ␤ isoform, which differ in their endogenous membrane-targeting properties and protein interactions (Buraei and Yang, 2010).…”

Section: Introductionmentioning

confidence: 99%

“…Alternative splicing of the exons encoding the vari-able regions gives rise to multiple variants of each ␤ isoform, which differ in their endogenous membrane-targeting properties and protein interactions (Buraei and Yang, 2010). These specific properties result in distinct stabilities of channel complexes (Campiglio et al, 2013), different subcellular targeting properties (Xie et al, 2007;Subramanyam et al, 2009;Obermair et al, 2010), and differential functional modulation of calcium currents (Helton and Horne, 2002). Nevertheless, the full complement of ␤ subunits expressed in the brain is still not completely known and our understanding of their specific targeting properties or their specific involvement in particular neuronal functions is still rudimentary.…”

Section: Introductionmentioning

confidence: 99%

Differential Neuronal Targeting of a New and Two Known Calcium Channel β₄Subunit Splice Variants Correlates with Their Regulation of Gene Expression

Etemad

Obermair

Bindreither³

et al. 2014

J. Neurosci.

View full text Add to dashboard Cite

The ␤ subunits of voltage-gated calcium channels regulate surface expression and gating of Ca V 1 and Ca V 2 ␣ 1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain ␤ subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of ␤ isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual ␤ variants in specific neuronal functions. In the present study, an alternatively spliced ␤ 4 subunit lacking the variable N terminus (␤ 4e ) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and Ca V 2.1 surface expression in neurons. Compared with the other two known full-length ␤ 4 variants (␤ 4a and ␤ 4b ), ␤ 4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of ␤ 4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (␤ 4 -null) mice individually reconstituted with ␤ 4a , ␤ 4b , and ␤ 4e . Notably, the number of genes regulated by each ␤ 4 splice variant correlated with the rank order of their nuclear-targeting properties (␤ 4b Ͼ ␤ 4a Ͼ ␤ 4e ). Together, these findings support isoform-specific functions of ␤ 4 splice variants in neurons, with ␤ 4b playing a dual role in channel modulation and gene regulation, whereas the newly detected ␤ 4e variant serves exclusively in calcium-channel-dependent functions.

show abstract

“…Primary antibodies were applied in PBS/BSA/Triton at 4°C overnight and detected by fluorochrome-conjugated secondary antibodies (15). For staining of surface-expressed HA-tagged Ca V 1.2 constructs, living neurons were incubated with the rat anti-HA antibody for 30 min at 37°C (42,43). Then the cultures were rinsed in Hanks' buffered saline solution, fixed for 10 min with pF, blocked with normal goat serum, and incubated with the secondary antibody for 1 h (15).…”

Section: Immunocytochemistrymentioning

confidence: 99%

Reciprocal Interactions Regulate Targeting of Calcium Channel β Subunits and Membrane Expression of α1 Subunits in Cultured Hippocampal Neurons

Obermair

Schlick

Biase

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Auxiliary ␤ subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca 2؉ channels in heterologous cells. In brain, all four ␤ isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of ␤ subunits and their role in membrane expression of Ca V 1.2 ␣ 1 subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous ␤ subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged ␤ subunits demonstrated that all ␤ subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca V 1.2, thus indicating a great promiscuity in ␣ 1 -␤ interactions. In contrast, restricted axonal targeting of ␤ 1 and weak colocalization of ␤ 4b with Ca V 1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged Ca V 1.2 was strongly enhanced by all ␤ subunits in an isoform-specific manner. Conversely, mutating the ␣-interaction domain of Ca V 1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a ␤ subunit with the ␣-interaction domain is absolutely essential for membrane expression of ␣ 1 subunits, as well as for the subcellular localization of ␤ subunits, which by themselves possess little or no targeting properties.Voltage-gated Ca 2ϩ channels (Ca V ) 3 provide key pathways for Ca 2ϩ entry into neurons and translate membrane depolarization into neurotransmitter secretion and gene regulation.Ca V s are composed of a pore-forming ␣ 1 subunit and the auxiliary ␣ 2 ␦ and ␤ subunits (1). Whereas the ␣ 1 subunits are responsible for voltage sensing and ion conduction, the auxiliary subunits have been implicated in membrane targeting and modulation of channel properties (for review see Ref.2). Presynaptic Ca V s regulate neurotransmitter release (3), and postsynaptic Ca V s activate the transcriptional regulators cAMP-response element-binding protein (CREB) and nuclear factor of activated T-cells (NFAT) (4, 5) and thus modulate long term potentiation (6). These functions reflect both the diversity of Ca V isoforms expressed in brain (7-11) and their differential subcellular localization in neurons (12-15).Four distinct ␤ isoforms have been identified (16 -19), all of which are expressed in brain (20 -23). They contain an Src homology 3 domain and a guanylate kinase domain (24 -27). However, the guanylate kinase fold is modified so that it can bind with high affinity to the so-called ␣-interaction domain (AID) in the intracellular I-II linker of Ca V ␣ 1 subunits (28, 29). The Src homology 3 and the guanylate kinase-like domains are highly conserved among the four genes encoding ␤ subunits (Cacnb1-b4; Fig. 1C), whereas t...

show abstract

Functional properties and modulation of extracellular epitope - tagged Ca_V2.1 voltage-gated calcium channels

Abstract: Depolarisation-induced Ca 2+ influx into electrically excitable cells is determined by the density of voltage-gated Ca

Cited by 26 publications

References 30 publications

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α ₂ δ-1 subunits

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α ₂ δ-1 subunits

Differential Neuronal Targeting of a New and Two Known Calcium Channel β₄Subunit Splice Variants Correlates with Their Regulation of Gene Expression

Reciprocal Interactions Regulate Targeting of Calcium Channel β Subunits and Membrane Expression of α1 Subunits in Cultured Hippocampal Neurons

Contact Info

Product

Resources

About

Functional properties and modulation of extracellular epitope - tagged CaV2.1 voltage-gated calcium channels

Abstract: Depolarisation-induced Ca 2+ influx into electrically excitable cells is determined by the density of voltage-gated Ca

Cited by 26 publications

References 30 publications

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α 2 δ-1 subunits

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α 2 δ-1 subunits

Differential Neuronal Targeting of a New and Two Known Calcium Channel β4Subunit Splice Variants Correlates with Their Regulation of Gene Expression

Reciprocal Interactions Regulate Targeting of Calcium Channel β Subunits and Membrane Expression of α1 Subunits in Cultured Hippocampal Neurons

Contact Info

Product

Resources

About

Functional properties and modulation of extracellular epitope - tagged Ca_V2.1 voltage-gated calcium channels

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α ₂ δ-1 subunits

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α ₂ δ-1 subunits

Differential Neuronal Targeting of a New and Two Known Calcium Channel β₄Subunit Splice Variants Correlates with Their Regulation of Gene Expression