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.2010 Aug 6;285(32):24968-76.
doi: 10.1074/jbc.M110.145813. Epub 2010 Jun 3.

Endogenous SNAP-25 regulates native voltage-gated calcium channels in glutamatergic neurons

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Endogenous SNAP-25 regulates native voltage-gated calcium channels in glutamatergic neurons

Steven B Condliffe et al. J Biol Chem..

Abstract

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25(+/-) mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.

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Figures

FIGURE 1.
FIGURE 1.
Reduced VGCC current density in glutamatergic compared with GABAergic neurons.A andB, representative whole cell VGCC inward Ba2+ currents (IBa) in response to 10-mV increment step depolarizations from −80 to 60 mV recorded from glutamatergic (A) and GABAergic (B) hippocampal neurons.C, ethidium bromide-stained gel of the PCR products from adult rat hippocampus (lane 1, positive control), recorded GABAergic neurons (lanes 2 and4), recorded glutamatergic neurons (lanes 3 and5), and a mock harvest (lane 6, negative control) amplified with primers specific for VGlut and VGAT.D, meanI-V relationships of peakIBa current density in glutamatergic (n = 11) and GABAergic (n = 9) neurons.E, representative current traces in response to a 1-s depolarization were normalized and aligned to compare the open-state inactivation of VGCC currents recorded from glutamatergic and GABAergic neurons.F, inactivation rate constants of VGCC currents in glutamatergic and GABAergic neurons evoked by the voltage protocol shown inA. Current traces were best fit with a double-exponential function where fast (τfast) and slow time constants (τslow) are represented as the mean ± S.E. (error bars) of 9 experiments (**,p ≤ 0.01).G, voltage-dependent activation curves of VGCC currents was elicited in glutamatergic (n = 8) and GABAergic (n = 8) neurons where tail currents generated by a repolarization to −40 mV were measured, normalized to the largest tail current in the series, and plotted against the prepulse voltage.H, voltage dependence of steady-state inactivation of VGCCs in glutamatergic (n = 7) and GABAergic (n = 8) neurons. A 4-s prepulse to the indicated potential was followed by a 10-ms test pulse to 0 mV, where test pulse currents for each prepulse were normalized to the peak current of the series.
FIGURE 2.
FIGURE 2.
Glutamatergic and GABAergic neurons exhibit a diverse sensitivity to specific VGCC subtype blockers.A, immunocytochemical staining of neuronal cultures for L- and P/Q-type channels. GABAergic neurons are revealed by double labeling for glutamic acid decarboxylase (GAD,green).Scale bar, 10 μm.B andC, representative traces of peakIBaversus time recorded from glutamatergic (B) and GABAergic (C) neurons during 50-ms depolarizations from a holding potential of −70 mV to −10 mV every 5 s with application of the specific VGCC blockers at the indicated times.D, inhibition of peakIBa by nifedipine (1 μm), ω-conotoxin-GVIA (1 μm), or ω-agatoxin-IVA (250 nm) in glutamatergic and GABAergic neurons expressed as the percentage ofIBa inhibited after administration of each specific blocker (n = 6–11).Error bars, S.E. of the mean.
FIGURE 3.
FIGURE 3.
Silencing of endogenous SNAP-25 in glutamatergic neurons augments VGCC properties.A, meanI-V relationships ofIBa current density in glutamatergic neurons co-transfected with pSuper SNAP-25 siRNA and GFP (n = 10), pSuper scrambled siRNA and GFP (n = 8), or GFP alone (n = 9).B, inhibition of peakIBa by nifedipine (1 μm), ω-conotoxin-GVIA (1 μm), or ω-agatoxin-IVA (250 nm) in glutamatergic neurons co-transfected with iSNAP-25 and GFP or in nontransfected glutamatergic neurons. Data are expressed as the percentage ofIBa inhibited after administration of each specific blocker (n = 6–12).C, voltage dependence of steady-state activation of isolated P/Q-type current in glutamatergic neurons co-transfected with pSuper SNAP-25 and GFP (n = 8) or GFP alone (n = 9).D, voltage dependence of steady-state inactivation of isolated P/Q-type current in glutamatergic neurons co-transfected with pSuper SNAP-25 and GFP (n = 8) or GFP alone (n = 9).Error bars, S.E. of the mean.
FIGURE 4.
FIGURE 4.
Distribution of P/Q-type channels and syntaxin in SNAP-25-silenced neurons.A, triple immunofluorescence labeling of hippocampal neurons for SNAP-25 (red), VGlut (green), and glutamic acid decarboxylase (GAD;blue).Arrow indicates a GAD-positive GABAergic neuron expressing lower levels of SNAP-25.B andC, hippocampal neurons co-transfected with iSNAP-25 and GFP and labeled for SNAP-25 (B) or syntaxin (C). Note the reduction of SNAP-25 expression (B) in the transfected cell whereas syntaxin expression (C) was not affected.D–F, labeling of iSNAP-25-GFP-transfected cultures for SNAP25 (green) and P/Q-type channels (red). Note that P/Q channels are expressed in the cell soma (D) and sorted in neurite varicosities (E) and growth cones (F) of both control and SNAP-25-silenced neurons. Neurons inF are triple-labeled for SNAP-25 (blue).Scale bars, 10 μm (A,B, andD), 25 μm (C), 7 μm (E), and 3.5 μm (F).
FIGURE 5.
FIGURE 5.
SNAP-25 exogenous expression in GABAergic neurons down-regulates VGCC function.A–D, immunolocalization of GABA in a SNAP-25-GFP-transfected neuron. Note the presence of SNAP-25 at the plasma membrane of the somatic region of transfected neurons (inset).E, meanI-V relationships ofIBa current density in GABAergic neurons transfected with either GFP (n = 8) or SNAP-25-GFP (n = 13).F, inhibition of peakIBa by nifedipine (1 μm), ω-conotoxin-GVIA (1 μm), or ω-agatoxin-IVA (250 nm) in GABAergic neurons transfected with SNAP-25-GFP or in nontransfected GABAergic neurons. Data are expressed as the percentage ofIBa inhibited after administration of each specific blocker (n = 6–12).WT, wild type.
FIGURE 6.
FIGURE 6.
VGCC properties in wild type and SNAP-25 heterozygous mouse neurons.A, meanI-V relationships ofIBa current density in wild type (n = 12) andSnap-25+/− (n = 12) glutamatergic neurons.B, meanI-V relationships ofIBa current density in wild type (n = 9) andSnap-25+/− (n = 9) GABAergic neurons.C, enrichment of Na+/K+-ATPase and SNAP-25 in membrane fractions prepared from mouse brains. The heat shock protein Hsp70 is recovered mainly in the cytosolic fraction.D, membrane fractions prepared from heterozygous samples contain reduced levels of SNAP-25 but comparable levels of the NMDA receptor subunits NR1 and NR2A, and syntaxin relative to wild type (wt). Note the equivalent expression of the CaV2.1 P/Q-type channels and the α2δ2 auxiliary subunit of VGCCs in wild type and heterozygous fractions.
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