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α2δ expression sets presynaptic calcium channel abundance and release probability.

Hoppa MB, Lana B, Margas W, Dolphin AC, Ryan TA - Nature (2012)

Bottom Line: First, α2δ subunits set synaptic VGCC abundance, as predicted from their chaperone-like function when expressed in non-neuronal cells.Expression of α2δ with an intact MIDAS motif leads to an 80% increase in release probability, while simultaneously protecting exocytosis from blockade by an intracellular Ca(2+) chelator. α2δs harbouring MIDAS site mutations still drive synaptic accumulation of VGCCs; however, they no longer change release probability or sensitivity to intracellular Ca(2+) chelators.Our data reveal dual functionality of these clinically important VGCC subunits, allowing synapses to make more efficient use of Ca(2+) entry to drive neurotransmitter release.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, New York 10023, USA.

ABSTRACT
Synaptic neurotransmitter release is driven by Ca(2+) influx through active zone voltage-gated calcium channels (VGCCs). Control of active zone VGCC abundance and function remains poorly understood. Here we show that a trafficking step probably sets synaptic VGCC levels in rats, because overexpression of the pore-forming α1(A) VGCC subunit fails to change synaptic VGCC abundance or function. α2δs are a family of glycosylphosphatidylinositol (GPI)-anchored VGCC-associated subunits that, in addition to being the target of the potent neuropathic analgesics gabapentin and pregabalin (α2δ-1 and α2δ-2), were also identified in a forward genetic screen for pain genes (α2δ-3). We show that these proteins confer powerful modulation of presynaptic function through two distinct molecular mechanisms. First, α2δ subunits set synaptic VGCC abundance, as predicted from their chaperone-like function when expressed in non-neuronal cells. Second, α2δs configure synaptic VGCCs to drive exocytosis through an extracellular metal ion-dependent adhesion site (MIDAS), a conserved set of amino acids within the predicted von Willebrand A domain of α2δ. Expression of α2δ with an intact MIDAS motif leads to an 80% increase in release probability, while simultaneously protecting exocytosis from blockade by an intracellular Ca(2+) chelator. α2δs harbouring MIDAS site mutations still drive synaptic accumulation of VGCCs; however, they no longer change release probability or sensitivity to intracellular Ca(2+) chelators. Our data reveal dual functionality of these clinically important VGCC subunits, allowing synapses to make more efficient use of Ca(2+) entry to drive neurotransmitter release.

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Increased expression of α2δ and β subunits leads to increased P/Q Ca2+ channel accumulation at synapsesa, Co-expression of vGmOr2 (left), eGFP–α1AE1656K (middle), overlay (right). b, Exocytic response for vGmOr2 alone (left; n=9) and vGmOr2 coexpressed with eGFP-α1AE1656K (right; n=6) reveals a significant toxin-resistant response (20±7.1% of pre-toxin). c, Average traces of single AP vGmOr2 responses ±eGFP-α1AE1656K (n>8). Arrow indicates stimulation with 1 AP. d, Average ΔF response for data in (c) (control 1.76±0.28; n=14; + eGFP–α1AE1656K 1.91±0.35; n=9; (p>0.1). e, Presynaptic α1A abundance. Green arrows indicate transfected boutons, white arrows indicate non-transfected immunopositive α1A channel puncta. f, ratio of α1A immunofluorescence intensity in transfected puncta compared to untransfected puncta (n≥8 cells for all conditions). All stated values are mean±SEM. Inset linear pseudo color LUT scale. Scale bar for all images = 4 μm.
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Figure 1: Increased expression of α2δ and β subunits leads to increased P/Q Ca2+ channel accumulation at synapsesa, Co-expression of vGmOr2 (left), eGFP–α1AE1656K (middle), overlay (right). b, Exocytic response for vGmOr2 alone (left; n=9) and vGmOr2 coexpressed with eGFP-α1AE1656K (right; n=6) reveals a significant toxin-resistant response (20±7.1% of pre-toxin). c, Average traces of single AP vGmOr2 responses ±eGFP-α1AE1656K (n>8). Arrow indicates stimulation with 1 AP. d, Average ΔF response for data in (c) (control 1.76±0.28; n=14; + eGFP–α1AE1656K 1.91±0.35; n=9; (p>0.1). e, Presynaptic α1A abundance. Green arrows indicate transfected boutons, white arrows indicate non-transfected immunopositive α1A channel puncta. f, ratio of α1A immunofluorescence intensity in transfected puncta compared to untransfected puncta (n≥8 cells for all conditions). All stated values are mean±SEM. Inset linear pseudo color LUT scale. Scale bar for all images = 4 μm.

Mentions: VGCCs are composed of pore-forming α1 and auxiliary β and α2δ subunits8,9. In central synapses neurotransmitter release is generally driven by P/Q-type (α1A) and/or N-type (α1B)10 VGCCs. Based on the failure of α1A overexpression to increase synaptic strength, it had been suggested that VGCCs functionally coupled to presynaptic release machinery is limited by a fixed number of available “slots” where channels can insert into the synaptic membrane11. We examined the existence of such a bottleneck by expressing eGFP-α1A12 together with a reporter of presynaptic exocytosis (vGlut1 with a luminal tag mOrange2, vGmOr2) and carried out retrospective immunocytochemistry to probe the abundance of α1A in transfected compared to control neurons. eGFP-α1A correctly trafficked to nerve terminals as it co-localized well with the vesicle-targeted reporter (Fig. 1a). In order to ensure eGFP-α1A functionally integrated with endogenous channels to drive neurotransmitter release we introduced a point mutation (E1656K), rendering this channel insensitive to the antagonist ω-agatoxin IVA13. Under control conditions a combination of ω-agatoxin IVA and the α1B inhibitor ω-conotoxin GVIA completely blocked vGmOr2 responses to action potential (AP) firing, however in the presence of eGFP-α1AE1656K a significant fraction of the response remains (Fig 1b). Measurements of single AP responses showed that expression of this exogenous α1A did not alter exocytosis efficiency compared to controls (Fig. 1c–d), consistent with the “slot” hypothesis11. However, retrospective immunocytochemistry using an anti-α1A antibody whose specificity was verified using shRNA-mediated α1A knockdown (Fig. S1) showed that transfected and control nerve terminals had similar immunoreactivity (Fig. 1e–f) while at the cell soma it had doubled (Fig S2). These results demonstrate that synaptic VGCC abundance is likely limited by trafficking from the cell soma and failure to increase synaptic performance does not result from a fixed number of active zone insertion sites. α2δ and β auxiliary VGCC subunits are both strong candidates for modulating such trafficking as they control functional expression of α1 subunits when co-expressed in non-neuronal cells14,15. We coexpressed individual auxiliary subunits with the reporter vGlut1-pHluorin (vGpH) in neurons and carried out measurements of exocytosis and immunocytochemistry as described above. These experiments demonstrated that expression of either α2δ-1 or β4 subunits led to a significant increase (~3-fold, p>0.05) in synaptic abundance of α1A (Fig. 1e–f). Similar results were obtained with overexpression of α2δ-2 (Fig. 1f). Furthermore, introduction of shRNA targeting α2δ-1 caused depletion of α1A at nerve terminals (Fig. 1e–f, Figure S3), while leaving the somatic concentration unaltered (data not shown). These results demonstrate that synaptic α1A levels are titrated by expression of auxiliary VGCC subunits.


α2δ expression sets presynaptic calcium channel abundance and release probability.

Hoppa MB, Lana B, Margas W, Dolphin AC, Ryan TA - Nature (2012)

Increased expression of α2δ and β subunits leads to increased P/Q Ca2+ channel accumulation at synapsesa, Co-expression of vGmOr2 (left), eGFP–α1AE1656K (middle), overlay (right). b, Exocytic response for vGmOr2 alone (left; n=9) and vGmOr2 coexpressed with eGFP-α1AE1656K (right; n=6) reveals a significant toxin-resistant response (20±7.1% of pre-toxin). c, Average traces of single AP vGmOr2 responses ±eGFP-α1AE1656K (n>8). Arrow indicates stimulation with 1 AP. d, Average ΔF response for data in (c) (control 1.76±0.28; n=14; + eGFP–α1AE1656K 1.91±0.35; n=9; (p>0.1). e, Presynaptic α1A abundance. Green arrows indicate transfected boutons, white arrows indicate non-transfected immunopositive α1A channel puncta. f, ratio of α1A immunofluorescence intensity in transfected puncta compared to untransfected puncta (n≥8 cells for all conditions). All stated values are mean±SEM. Inset linear pseudo color LUT scale. Scale bar for all images = 4 μm.
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Figure 1: Increased expression of α2δ and β subunits leads to increased P/Q Ca2+ channel accumulation at synapsesa, Co-expression of vGmOr2 (left), eGFP–α1AE1656K (middle), overlay (right). b, Exocytic response for vGmOr2 alone (left; n=9) and vGmOr2 coexpressed with eGFP-α1AE1656K (right; n=6) reveals a significant toxin-resistant response (20±7.1% of pre-toxin). c, Average traces of single AP vGmOr2 responses ±eGFP-α1AE1656K (n>8). Arrow indicates stimulation with 1 AP. d, Average ΔF response for data in (c) (control 1.76±0.28; n=14; + eGFP–α1AE1656K 1.91±0.35; n=9; (p>0.1). e, Presynaptic α1A abundance. Green arrows indicate transfected boutons, white arrows indicate non-transfected immunopositive α1A channel puncta. f, ratio of α1A immunofluorescence intensity in transfected puncta compared to untransfected puncta (n≥8 cells for all conditions). All stated values are mean±SEM. Inset linear pseudo color LUT scale. Scale bar for all images = 4 μm.
Mentions: VGCCs are composed of pore-forming α1 and auxiliary β and α2δ subunits8,9. In central synapses neurotransmitter release is generally driven by P/Q-type (α1A) and/or N-type (α1B)10 VGCCs. Based on the failure of α1A overexpression to increase synaptic strength, it had been suggested that VGCCs functionally coupled to presynaptic release machinery is limited by a fixed number of available “slots” where channels can insert into the synaptic membrane11. We examined the existence of such a bottleneck by expressing eGFP-α1A12 together with a reporter of presynaptic exocytosis (vGlut1 with a luminal tag mOrange2, vGmOr2) and carried out retrospective immunocytochemistry to probe the abundance of α1A in transfected compared to control neurons. eGFP-α1A correctly trafficked to nerve terminals as it co-localized well with the vesicle-targeted reporter (Fig. 1a). In order to ensure eGFP-α1A functionally integrated with endogenous channels to drive neurotransmitter release we introduced a point mutation (E1656K), rendering this channel insensitive to the antagonist ω-agatoxin IVA13. Under control conditions a combination of ω-agatoxin IVA and the α1B inhibitor ω-conotoxin GVIA completely blocked vGmOr2 responses to action potential (AP) firing, however in the presence of eGFP-α1AE1656K a significant fraction of the response remains (Fig 1b). Measurements of single AP responses showed that expression of this exogenous α1A did not alter exocytosis efficiency compared to controls (Fig. 1c–d), consistent with the “slot” hypothesis11. However, retrospective immunocytochemistry using an anti-α1A antibody whose specificity was verified using shRNA-mediated α1A knockdown (Fig. S1) showed that transfected and control nerve terminals had similar immunoreactivity (Fig. 1e–f) while at the cell soma it had doubled (Fig S2). These results demonstrate that synaptic VGCC abundance is likely limited by trafficking from the cell soma and failure to increase synaptic performance does not result from a fixed number of active zone insertion sites. α2δ and β auxiliary VGCC subunits are both strong candidates for modulating such trafficking as they control functional expression of α1 subunits when co-expressed in non-neuronal cells14,15. We coexpressed individual auxiliary subunits with the reporter vGlut1-pHluorin (vGpH) in neurons and carried out measurements of exocytosis and immunocytochemistry as described above. These experiments demonstrated that expression of either α2δ-1 or β4 subunits led to a significant increase (~3-fold, p>0.05) in synaptic abundance of α1A (Fig. 1e–f). Similar results were obtained with overexpression of α2δ-2 (Fig. 1f). Furthermore, introduction of shRNA targeting α2δ-1 caused depletion of α1A at nerve terminals (Fig. 1e–f, Figure S3), while leaving the somatic concentration unaltered (data not shown). These results demonstrate that synaptic α1A levels are titrated by expression of auxiliary VGCC subunits.

Bottom Line: First, α2δ subunits set synaptic VGCC abundance, as predicted from their chaperone-like function when expressed in non-neuronal cells.Expression of α2δ with an intact MIDAS motif leads to an 80% increase in release probability, while simultaneously protecting exocytosis from blockade by an intracellular Ca(2+) chelator. α2δs harbouring MIDAS site mutations still drive synaptic accumulation of VGCCs; however, they no longer change release probability or sensitivity to intracellular Ca(2+) chelators.Our data reveal dual functionality of these clinically important VGCC subunits, allowing synapses to make more efficient use of Ca(2+) entry to drive neurotransmitter release.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, New York 10023, USA.

ABSTRACT
Synaptic neurotransmitter release is driven by Ca(2+) influx through active zone voltage-gated calcium channels (VGCCs). Control of active zone VGCC abundance and function remains poorly understood. Here we show that a trafficking step probably sets synaptic VGCC levels in rats, because overexpression of the pore-forming α1(A) VGCC subunit fails to change synaptic VGCC abundance or function. α2δs are a family of glycosylphosphatidylinositol (GPI)-anchored VGCC-associated subunits that, in addition to being the target of the potent neuropathic analgesics gabapentin and pregabalin (α2δ-1 and α2δ-2), were also identified in a forward genetic screen for pain genes (α2δ-3). We show that these proteins confer powerful modulation of presynaptic function through two distinct molecular mechanisms. First, α2δ subunits set synaptic VGCC abundance, as predicted from their chaperone-like function when expressed in non-neuronal cells. Second, α2δs configure synaptic VGCCs to drive exocytosis through an extracellular metal ion-dependent adhesion site (MIDAS), a conserved set of amino acids within the predicted von Willebrand A domain of α2δ. Expression of α2δ with an intact MIDAS motif leads to an 80% increase in release probability, while simultaneously protecting exocytosis from blockade by an intracellular Ca(2+) chelator. α2δs harbouring MIDAS site mutations still drive synaptic accumulation of VGCCs; however, they no longer change release probability or sensitivity to intracellular Ca(2+) chelators. Our data reveal dual functionality of these clinically important VGCC subunits, allowing synapses to make more efficient use of Ca(2+) entry to drive neurotransmitter release.

Show MeSH
Related in: MedlinePlus