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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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α2δ MIDAS motif is essential for coupling Ca2+ channels to exocytosisa,Top: Presynaptic α1A abundance. Green arrows indicate transfected boutons, white arrows indicate non-transfected immunopositive α1A channel puncta.. Scale bar = 2 μm. Bottom: Ratio of α1A staining in synaptic boutons. Dashed lines represent ratios taken from Fig. 1f as indicated. b, Top: Representative vGpH responses to 1 AP (arrow) as a fraction of the measured RRP Bottom: vGpH and α2δ-1 MIDASAAA (Pv=0.33±0.017) compared to data from Fig. 2f as indicated. c, Top: Representative responses to 1 AP-driven Ca2+ influx (Fluo5F 3F/F). Bottom: Peak 1 AP Fluo 5F ΔF/F values in cells co-transfected with VAMPmCh (n=11) and α2δ-1 MIDASAAA (0.88±0.1; n=6) normalized to VAMPmCh alone (*p<0.05). d,Top: Representative vGpH response to 1 AP in a neuron co-expressing α2δ-1 MIDASAAA as indicated. Bottom: Resistance to EGTA block (% block = 51±5, p=0.63) dashed lines compare data from Fig. 3e as indicated. e, Representative vGpH responses to 1 AP. f, 1 AP response (%NH4Cl): vGpH=1.65±0.17; vGpH+α2δ-1shRNA=0.4±0.08, v G p H +α2δ-1shRNA+α2δ-2=3.10±.53; vGpH+α2δ-1shRNA+α2δ-2 MIDASAAA=1.02±.41; vGpH+α2δ-2=2.44±0.36. Values are mean±SEM, *p<0.01, n≥7, (*p<0.05).
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Figure 4: α2δ MIDAS motif is essential for coupling Ca2+ channels to exocytosisa,Top: Presynaptic α1A abundance. Green arrows indicate transfected boutons, white arrows indicate non-transfected immunopositive α1A channel puncta.. Scale bar = 2 μm. Bottom: Ratio of α1A staining in synaptic boutons. Dashed lines represent ratios taken from Fig. 1f as indicated. b, Top: Representative vGpH responses to 1 AP (arrow) as a fraction of the measured RRP Bottom: vGpH and α2δ-1 MIDASAAA (Pv=0.33±0.017) compared to data from Fig. 2f as indicated. c, Top: Representative responses to 1 AP-driven Ca2+ influx (Fluo5F 3F/F). Bottom: Peak 1 AP Fluo 5F ΔF/F values in cells co-transfected with VAMPmCh (n=11) and α2δ-1 MIDASAAA (0.88±0.1; n=6) normalized to VAMPmCh alone (*p<0.05). d,Top: Representative vGpH response to 1 AP in a neuron co-expressing α2δ-1 MIDASAAA as indicated. Bottom: Resistance to EGTA block (% block = 51±5, p=0.63) dashed lines compare data from Fig. 3e as indicated. e, Representative vGpH responses to 1 AP. f, 1 AP response (%NH4Cl): vGpH=1.65±0.17; vGpH+α2δ-1shRNA=0.4±0.08, v G p H +α2δ-1shRNA+α2δ-2=3.10±.53; vGpH+α2δ-1shRNA+α2δ-2 MIDASAAA=1.02±.41; vGpH+α2δ-2=2.44±0.36. Values are mean±SEM, *p<0.01, n≥7, (*p<0.05).

Mentions: The finding that α2δ subunits form GPI-anchored proteins3 implies that their ability to change VGCC-exocytosis coupling is likely conveyed through an extracellular interaction. One possible candidate for exerting such influence lies in the highly-conserved VWA domain within α2δ22. A characteristic feature of this domain is its ability to interact with adhesion proteins via the MIDAS motif by sharing coordination of a divalent cation23–25. To examine the role of α2δ’s MIDAS motif we mutated three of five conserved key metal coordinating residues within the MIDAS motif to alanine22 and expressed the mutant protein (α2δ-1 MIDASAAA) in neurons together with functional reporters. α2δ-1 MIDASAAA was similar to wild type α2δ-1 in its ability to drive α1A accumulation at synapses (Fig. 4a). However, measurements of exocytosis from α2δ-1 MIDAS-mutants showed no enhancement of Pv (Fig. 4b), normal Ca2+ influx (Fig. 4c) and normal sensitivity to EGTA block of exocytosis (Fig. 4d). Furthermore α2δ-2 MIDASAAA, unlike intact α2δ-2, was unable to rescue the decrease in exocytosis resulting from shRNA-mediated α2δ-1 depletion (Fig. 4e,f). These data are consistent with the ability of this mutation to block enhancement of Ca2+ currents when expressed in heterologous systems22 (Fig. S8), but show that they do not prevent endogenous α2δ from functioning. Taken together, these results demonstrate that α2δ exerts its powerful control of synaptic VGCC function through at least two separate molecular mechanisms: a forward trafficking-step from cell body to presynaptic terminal that is independent of MIDAS motif integrity; and a local MIDAS-dependent interaction critical for proper VGCC function and coupling to exocytosis.


α2δ expression sets presynaptic calcium channel abundance and release probability.

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

α2δ MIDAS motif is essential for coupling Ca2+ channels to exocytosisa,Top: Presynaptic α1A abundance. Green arrows indicate transfected boutons, white arrows indicate non-transfected immunopositive α1A channel puncta.. Scale bar = 2 μm. Bottom: Ratio of α1A staining in synaptic boutons. Dashed lines represent ratios taken from Fig. 1f as indicated. b, Top: Representative vGpH responses to 1 AP (arrow) as a fraction of the measured RRP Bottom: vGpH and α2δ-1 MIDASAAA (Pv=0.33±0.017) compared to data from Fig. 2f as indicated. c, Top: Representative responses to 1 AP-driven Ca2+ influx (Fluo5F 3F/F). Bottom: Peak 1 AP Fluo 5F ΔF/F values in cells co-transfected with VAMPmCh (n=11) and α2δ-1 MIDASAAA (0.88±0.1; n=6) normalized to VAMPmCh alone (*p<0.05). d,Top: Representative vGpH response to 1 AP in a neuron co-expressing α2δ-1 MIDASAAA as indicated. Bottom: Resistance to EGTA block (% block = 51±5, p=0.63) dashed lines compare data from Fig. 3e as indicated. e, Representative vGpH responses to 1 AP. f, 1 AP response (%NH4Cl): vGpH=1.65±0.17; vGpH+α2δ-1shRNA=0.4±0.08, v G p H +α2δ-1shRNA+α2δ-2=3.10±.53; vGpH+α2δ-1shRNA+α2δ-2 MIDASAAA=1.02±.41; vGpH+α2δ-2=2.44±0.36. Values are mean±SEM, *p<0.01, n≥7, (*p<0.05).
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Figure 4: α2δ MIDAS motif is essential for coupling Ca2+ channels to exocytosisa,Top: Presynaptic α1A abundance. Green arrows indicate transfected boutons, white arrows indicate non-transfected immunopositive α1A channel puncta.. Scale bar = 2 μm. Bottom: Ratio of α1A staining in synaptic boutons. Dashed lines represent ratios taken from Fig. 1f as indicated. b, Top: Representative vGpH responses to 1 AP (arrow) as a fraction of the measured RRP Bottom: vGpH and α2δ-1 MIDASAAA (Pv=0.33±0.017) compared to data from Fig. 2f as indicated. c, Top: Representative responses to 1 AP-driven Ca2+ influx (Fluo5F 3F/F). Bottom: Peak 1 AP Fluo 5F ΔF/F values in cells co-transfected with VAMPmCh (n=11) and α2δ-1 MIDASAAA (0.88±0.1; n=6) normalized to VAMPmCh alone (*p<0.05). d,Top: Representative vGpH response to 1 AP in a neuron co-expressing α2δ-1 MIDASAAA as indicated. Bottom: Resistance to EGTA block (% block = 51±5, p=0.63) dashed lines compare data from Fig. 3e as indicated. e, Representative vGpH responses to 1 AP. f, 1 AP response (%NH4Cl): vGpH=1.65±0.17; vGpH+α2δ-1shRNA=0.4±0.08, v G p H +α2δ-1shRNA+α2δ-2=3.10±.53; vGpH+α2δ-1shRNA+α2δ-2 MIDASAAA=1.02±.41; vGpH+α2δ-2=2.44±0.36. Values are mean±SEM, *p<0.01, n≥7, (*p<0.05).
Mentions: The finding that α2δ subunits form GPI-anchored proteins3 implies that their ability to change VGCC-exocytosis coupling is likely conveyed through an extracellular interaction. One possible candidate for exerting such influence lies in the highly-conserved VWA domain within α2δ22. A characteristic feature of this domain is its ability to interact with adhesion proteins via the MIDAS motif by sharing coordination of a divalent cation23–25. To examine the role of α2δ’s MIDAS motif we mutated three of five conserved key metal coordinating residues within the MIDAS motif to alanine22 and expressed the mutant protein (α2δ-1 MIDASAAA) in neurons together with functional reporters. α2δ-1 MIDASAAA was similar to wild type α2δ-1 in its ability to drive α1A accumulation at synapses (Fig. 4a). However, measurements of exocytosis from α2δ-1 MIDAS-mutants showed no enhancement of Pv (Fig. 4b), normal Ca2+ influx (Fig. 4c) and normal sensitivity to EGTA block of exocytosis (Fig. 4d). Furthermore α2δ-2 MIDASAAA, unlike intact α2δ-2, was unable to rescue the decrease in exocytosis resulting from shRNA-mediated α2δ-1 depletion (Fig. 4e,f). These data are consistent with the ability of this mutation to block enhancement of Ca2+ currents when expressed in heterologous systems22 (Fig. S8), but show that they do not prevent endogenous α2δ from functioning. Taken together, these results demonstrate that α2δ exerts its powerful control of synaptic VGCC function through at least two separate molecular mechanisms: a forward trafficking-step from cell body to presynaptic terminal that is independent of MIDAS motif integrity; and a local MIDAS-dependent interaction critical for proper VGCC function and coupling to exocytosis.

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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Related in: MedlinePlus