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J. Biol. Chem., Vol. 275, Issue 48, 37807-37814, December 1, 2000

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Synaptic Localization and Presynaptic Function of Calcium Channel ₄-Subunits in Cultured Hippocampal Neurons^*

Silke Wittemann, Melanie D. Mark, Jens Rettig^§, and Stefan Herlitze^¶

From the  Department of Physiology II, University of Tuebingen, Ob dem Himmelreich 7, 72074 Tuebingen, Germany and the ^§ Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Goettingen, Germany

Received for publication, May 30, 2000, and in revised form, August 1, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Neurotransmitter release is triggered by the influx of Ca²⁺ into the presynaptic terminal through voltage gated Ca²⁺-channels. The shape of the presynaptic Ca²⁺ signal largely determines the amount of released quanta and thus the size of the synaptic response. Ca²⁺-channel function is modulated in particular by the auxiliary -subunits that interact intracellularly with the pore-forming ₁-subunit. Using retrovirus-mediated gene transfer in cultured hippocampal neurons, we demonstrate that functional GFP-₄ constructs colocalize with the synaptic vesicle marker synaptobrevin II and endogenous P/Q-type channels, indicating that ₄-subunits are localized to synaptic sites. Costaining with the dendritic marker MAP2 revealed that the ₄-subunit is transported to dendrites as well as axons. The nonconserved amino- and carboxyl-termini of the ₄-subunit were found to target the protein to the synapse. Physiological measurements in autaptic hippocampal neurons infected with green fluorescent protein (GFP)-₄ revealed an increase in both excitatory post-synaptic current amplitude and paired pulse facilitation ratio, whereas the GFP-₄ mutant, GFP-₄(50-407), which demonstrated a cytosolic localization pattern, did not alter these synaptic properties. In summary, our data suggest a pre-synaptic function of the Ca²⁺-channel ₄-subunit in synaptic transmission.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺-channels mediate voltage-dependent Ca²⁺-influx in subcellular compartments of neurons, triggering such diverse processes as neurotransmitter release and excitation-transcription coupling (1, 2). Neuronal Ca²⁺-channels consist of a pore-forming ₁-subunit and several auxiliary subunits (-, ₂-, and presumably -subunits), which are associated with the ₁-subunit. Ca²⁺-channel function is determined by different -subunits, which modify the gating properties of the channel and most likely the transport of the ₁-subunit to the cell surface (3-7). Expression of the four different -subunits has been shown in various brain regions such as the cerebellum and hippocampus. Here, -subunits were expressed in neuronal cell bodies, dendrites, and neuropils (8-11). A pre- and post-synaptic localization has been suggested for ₁-, ₃-, and ₄-subunits, but a precise role for any -subunit in synaptic transmission has not been described so far.

New approaches in cell biology to study protein targeting within a cell, e.g. GFP¹ and viral transfection methods, allow the overexpression of ion channel subunits in cultured neuronal cells to analyze their transport to specific subcellular compartments (12). Applying these methods, we investigated the distribution and specific function of the Ca²⁺-channel ₄-subunit in cultured hippocampal neurons. We demonstrated that the ₄-subunit is localized to presynaptic terminals and that its N and C termini are responsible for this specific targeting. Furthermore, we show that ₄-subunits of voltage-gated Ca²⁺-channels play an important physiological role in synaptic transmission by altering amplitude and activity-dependent properties of the synaptic response.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of GFP Fusion Proteins and Deletion Mutants-- The rat ₄ cDNA (13) was amplified with the following primer pairs, which include restriction sites for subcloning: 5'-GATCTCGAGATGTCGTCCTCCTACGCCAAG and 3'-ACGGTCGACTCAAAGCCTATGTCGGGAGTC.

The constructs were subcloned in-frame into pEGFP-C₃ (CLONTECH) and then excised for cloning into the Semliki Forest virus vector pSFV1 (Life Technologies, Inc.). Deletion mutants were constructed using a single PCR reaction where restriction sites for subcloning into pEGFP-C₃ (CLONTECH) were placed within the oligonucleotide primer. The following primer pairs were used for the deletion mutants: N terminus (aa 1-49), 5'-GATCTCGAGATGTCGTCCTCCTACGCCAAG and 3'-GGGGGATCCCCCCTGTCTGAGGATGAAGCTGGT; C terminus (aa 408-519), 5'-CCCAAGCTTCCTATGACCCCATTGCTGGGG and 3'-ACGGTCGACTCAAAGCCTATGTCGGGAGTC; 50-407, 5'-GATCTCGAGTCAGCAGATTCCTATACA and 3'-CCCAAGCTTGGTGCTACTGCTTGTGTGGGT.

The pEGFP-C₃-₄ deletion clones were subcloned into pSFV by blunt end cloning and correct orientation of the constructs were verified by cDNA sequencing. cDNAs encoding ₄ (13), GFP-₄, and GFP-₄(50-407) in pEGFP-C₃ (CLONTECH) were used for whole cell recordings. Western blotting of infected HEK293 cells with a monoclonal anti-GFP antibody (CLONTECH) was performed according to standard procedures as described by Mark et al. (14).

Cell Culture and Electrophysiology-- HEK293 cells were transfected with the Ca²⁺-channel subunits _1A, ₂, and ₄, GFP-₄, or GFP-₄(50-407). cDNAs and whole cell recordings were performed as published previously (15). Membrane capacitance and series resistance were compensated electronically using the patch clamp amplifier (EPC-9; HEKA, Lambrecht, Germany). Voltage protocol design and data acquisition were performed using Pulse++, version 1.7 software (Ulix GmbH, Tuebingen, Germany) on a Macintosh Power PC. Inactivation measurements for control of functional GFP constructs (Figs. 1 and 5) were performed with 10 mM Ba²⁺ as the current carrier at room temperature. 2- and 10-ms inactivation protocols were measured with 4 mM Ca²⁺ instead of Ba²⁺ as the current carrier, also at room temperature (extracellular solution in mM: 100 Tris, 4 MgCl₂, 4 CaCl₂, pH 7.3, with methanesulfonic acid). Ca²⁺- and Ba²⁺-currents were analyzed using the IGOR data analysis package (WaveMetrics, Lake Oswego, OR). Peak currents were determined from the current measured at +10 mV. Currents were elicited by a 500-ms voltage ramp from 70 mV to +50 mV. Time constants of inactivation (-inact) were determined by fitting the decay phase of a 1000-ms test pulse from 70 mV to +10 mV with a single exponential or, as shown in Fig. 9, by fitting the decreasing tail currents elicited by 2- or 10-ms test pulses from 70 mV to +10 mV every 50 ms with a single exponential. The percent current reduction was determined by comparing the size of the first elicited tail current and the last elicited tail current with the 20 Hz stimulation protocol. 20 test pulses were applied within the 20 Hz stimulation protocol in HEK293 cells. Statistical significance was expressed as ** p < 0.01. All error values (±) and bars in this publication are S.E.s.

Micro-island cultures of hippocampal neurons were prepared according to a modified version of published procedures (16). After 9-14 days in culture, cells were infected with 50 µl of an activated Semliki Forest virus containing the cDNAs of GFP-₄ or GFP-₄-deletion constructs following a protocol given in Ashery et al. (17). All measurements were performed 6-18 h after infection. Only dots containing a single neuron forming excitatory synapses (autapses) were used. Extracellular recording solution contained (in mM): 172 NaCl, 2.4 KCl, 10 HEPES, 10 glucose, 4 CaCl₂, 4 MgCl₂, and 0.03 CdCl₂ (pH 7.3, 350 mosmol). Patch pipettes (2-3 megaohms) were pulled from borosilicate glass (TWF 150, World Precision Instruments) on a Sutter puller and back-filled with the following (in mM): 145 potassium gluconate, 15 HEPES, 1 potassium-EGTA, 4 Na-ATP, 0.4 Na-GTP (pH 7.3). Currents were recorded and analyzed as published previously by Lao et al. (18).

Immunocytochemistry-- Continental hippocampal cultures were prepared and infected as described above and fixed as described by Mark et al. (14). Neurons were incubated with synaptobrevin II antibody (C69.1 at 1:1000, a gift from R. Jahn, Göttingen, Germany), Ca²⁺-channel _1A-subunit antibody (Chemicon; 1:60) or MAP2 antibody (Sigma; 1:1000) and then with an Alexa 546-coupled secondary antibody (Molecular Probes, Leiden, Netherlands). Cells were embedded in Fluoromount (133 mM Tris-HCl, 30% glycerol, 11% Mowiol, 2% diazabicyclo(2.2.2)octane. Fluorescence was detected with a conventional fluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany), and images were analyzed with the Metamorph imaging system.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A fusion construct between GFP and the ₄-subunit was generated to analyze the distribution and function of this Ca²⁺-channel subunit (Fig. 1a). The GFP was tagged in frame to the N terminus of the ₄-subunit using the mammalian expression vector pEGFP-C₃ and subcloned into the Semliki Forest virus vector (pSFV). We first investigated whether the fusion construct of the virus expresses a protein of the correct size. Western blot analysis of HEK293 cells infected with GFP-₄ revealed an 83-kDa band, the predicted size of the GFP-₄ fusion protein (Fig. 1b). In addition, the GFP-₄ protein assembled with the _1A- and ₂-subunits to form a functional voltage-gated Ca²⁺-channel in HEK293 cells. Whole cell peak currents were measured at +10 mV during a 500-ms voltage ramp from 70 to +50 mV and increased from 38 ± 14 pA (n = 7) for _1A/₂ to 602 ± 314 pA (n = 11) with the GFP-₄ fusion protein, which was comparable to the wild-type ₄ currents (-744 ± 209 pA (n = 23)) (Fig. 1c). In addition, GFP-₄ revealed the characteristic slow inactivation time constants at 0, +5, +10, and +15 mV (-inact) as described for wild-type ₄-subunit assembled with P/Q-type Ca²⁺-channels (188 ± 22 (0 mV), 145 ± 18 (+5 mV), 131 ± 18 (+10 mV), and 130 ± 15 ms (+15 mV) (n = 6) for wild-type-₄; (178 ± 29 (0 mV), 149 ± 18 (+5 mV), 136 ± 12 (+10 mV), and 147 ± 22 ms (+15 mV) (n = 6) for GFP-₄) (Fig. 1d) (19). Thus, GFP-₄ constructs yield functional ₄-protein, which alters the electrophysiological characteristics of the P/Q-type Ca²⁺-channels in the expected manner.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Western blot analysis and whole cell recordings of HEK293 cells expressing GFP-4. a, schematic representation of the GFP-4 fusion construct used in the experiments described. GFP was tagged in-frame to the N terminus of 4. The 4-subunit consists of 519 aa. b, Western blot analysis of GFP and GFP-4 expressed in HEK293 cells. Proteins were detected with a monoclonal antibody against GFP. Purified GFP-4 protein shows the expected size of 83 kDa. c, whole cell recordings of HEK293 cells cotransfected with the 1A, 2, and GFP-4 or wild-type-4. Left, representative Ba2+-currents from an 1A, 2, and GFP-4 assembled channels were elicited by 4-ms, 5-mV step potentials from 50 to +50 mV from a holding potential of 70 mV. Right, Ba2+-currents were measured at +10 mV during the 500-ms voltage ramps. Bars represent cells transfected with the following DNAs: black bar, 1A, 2, and GFP; white bar, 1A, 2, wild-type-4, and GFP; gray bar, 1A, 2, and GFP-4. The Ba2+-currents are significantly larger for cells expressing the 4-subunit (p > 0.01, two-tailed t test) but are not significantly different between wild-type4 and GFP-4. d, left, representative traces of Ba2+-currents from 1A, 2, and GFP-4 assembled channels elicited by a 1000-ms test pulse to 50, 0, +5, +10, and +15 mV from a holding potential of 70 mV. Right, time constants for inactivation curves (-inact in ms) measured at 0, +5, +10, and +15 mV. Values for 1A, 2, and wild-type 4 (open circles) or GFP-4 (filled diamonds) are not significantly different.

To investigate the distribution of ₄ in hippocampal neurons, 9-14-day-old neurons were infected with the GFP-₄ construct using the Semliki Forest virus gene expression system. Overexpressed GFP-₄-subunits displayed a punctate staining pattern along the neuronal processes (Fig. 2, a-c), as observed for vesicle-transported proteins like synaptobrevin II, syntaxin, or syntaphilin, whereas overexpressed GFP alone showed a diffuse, cytosolic distribution along the neurites (Fig. 2, d-f). Colocalization with the synaptic vesicle marker synaptobrevin II (Fig. 2c) revealed that GFP-₄ was indeed transported to synaptic sites. P/Q-type Ca²⁺-channels are highly expressed in presynaptic terminals (20) and play a fundamental role in synaptic transmission (2). Because ₄-subunits can assemble with _1A-subunits to form functional channels in HEK293 cells (Fig. 1), we investigated whether ₄-subunits colocalize with endogenous presynaptic Ca²⁺-channels in hippocampal neurons. Costaining between the _1A-subunit encoding for the P/Q-type channel and GFP-₄ demonstrated the colocalization of both proteins (Fig. 3). Next, we investigated whether the ₄-subunit is localized to dendrites or axons. Double-labeling with the dendritic marker MAP2 identified the fact that GFP-₄ is transported to both dendrites and axons (Fig. 4). Thus, our results highly suggest that the ₄-subunit is targeted to synaptic sites.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Colocalization of GFP-4 with synaptobrevin II in cultured hippocampal neurons. a, fluorescence patterns of neurons from low density hippocampal cultures infected with GFP-4 reveal a punctate staining. b, hippocampal cells were stained with an anti-synaptobrevin II antibody and visualized with an Alexa 546-coupled secondary antibody. A punctate staining pattern similar to that seen with GFP-4 was observed. c, overlay of a and b in the indicated area demonstrates that GFP-4 is partially colocalized with the synaptic vesicle marker synaptobrevin II (yellow). d, fluorescence pattern of neurons from low density hippocampal cultures infected with GFP alone demonstrates a diffuse, cytosolic staining. e, hippocampal cells were stained with an anti-synaptobrevin II antibody and visualized with an Alexa 546-coupled secondary antibody. f, overlay of d and e in the indicated area demonstrates that GFP is not colocalized with the synaptic vesicle marker synaptobrevin II (yellow). Scale bars in a, b, d, and e =10 µm and in c and f = 2 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Colocalization of GFP-4 with Ca2+-channel 1A-subunit in cultured hippocampal neurons. a, fluorescence patterns of neurons from low density hippocampal cultures infected with GFP-4 reveal a punctate staining. b, hippocampal cells were stained with an anti-1A antibody and visualized with an Alexa 546-coupled secondary antibody. A punctate staining pattern similar to that seen with GFP-4 was observed. c, overlay of a and b in the indicated area demonstrates that GFP-4 is partially colocalized with the presynaptic P/Q-type Ca2+-channel subunit 1A (yellow). Scale bars in a and b = 10 µm and in c = 5 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 4.   Localization of GFP-4 and MAP2 in cultured hippocampal neurons. a, fluorescence patterns of neurons from low density hippocampal cultures infected with GFP-4 reveal staining in dendrites and axons. b, hippocampal cells were stained with an anti-MAP2 antibody and visualized with an Alexa 546-coupled secondary antibody. MAP2 stains only the dendrites and not the axon. Scale bars = 10 µm.

Based on the sequence alignment, the predicted structure of _1b-subunit, and functional analysis, -subunits can be divided into five domains (21, 22) (Fig. 5a). A high degree of conservation among the -subunits was observed for domains II and IV, but not for domains I (N terminus), III, and V (C terminus). To determine which domain of ₄ is responsible for targeting this subunit to its specific subcellular localization, we produced several deletion constructs of GFP-₄ according to the domain assignment (Fig. 5a). These constructs expressed fusion proteins of the expected size (Fig. 5b). The minimal segments required for punctate localization and colocalization with synaptobrevin II were domain I (N terminus, aa 1-49) and domain V (C terminus, aa 408-519) of the ₄-subunit. 97% of the cells infected with the C terminus and 72% of the cells infected with the N terminus colocalized with synaptobrevin II and showed a punctate pattern (Table I). Thus, either N or C terminus alone is sufficient for colocalization with synaptobrevin II (Fig. 6, a-f). In contrast, a deletion construct lacking both the N and C termini (aa 50-407; domains II-IV), such as GFP alone, revealed a homogenous, cytosolic localization. 72% of these cells showed no synaptobrevin II colocalization (Fig. 6, g-i) (data summarized in Table I). The punctate staining pattern and its colocalization with synaptobrevin II indicates that the N and C termini of GFP-₄ are sufficient for synaptic targeting.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   GFP-4 deletion constructs. a, schematic representation of 4 domain structure and derived deletion constructs. The -subunit is divided into 5 domains (I, II, III, IV, V). The black bars represent the domains with a high degree of conservation between all four -subunit isoforms (1b, 3, 2a, and 4), and the gray bars represent domains with a low degree of conservation (22, 41). b, Western blot analysis of GFP and GFP-4 deletion constructs expressed in HEK293 cells. Proteins were detected as described in Fig. 1b legend. c, whole cell recordings of HEK293cells cotransfected with 1A, 2, and with or without GFP-4(50-407). Left, representative Ba2+-currents from 1A, 2, and GFP-4(50-407) assembled channels were elicited by 4-ms, 5-mV step potentials from 50 to +50 mV from a holding potential of 70 mV. Right, Ba2+-currents were measured at +10 mV during 500-ms voltage ramps. Bars represent cells transfected with the following DNAs: black bar, 1A, 2, and GFP; striped bar, 1A, 2, and GFP-4(50-407). The Ba2+-currents are significantly larger for cells expressing the GFP-4(50-407) construct (p > 0.01, two-tailed t test). d, left, representative traces of a Ba2+-currents from 1A, 2, and GFP-4(50-407) assembled channels elicited by a 1000-ms test pulse to 50, 0, +5, +10, and +15 mV from a holding potential of 70 mV. Right, time constants for inactivation curves (-inact in ms) measured at 0, +5, +10, and +15 mV. Values for Ca2+-channels assembled with the GFP-4 (open circles) or GFP-4(50-407) (filled squares) are not significantly different.

View this table: [in this window] [in a new window]   Table I Statistical analysis of punctate staining pattern in hippocampal neurons infected with various 4 constructs Low density hippocampal cultures were prepared and infected with various 4 constructs after 9-14 days in culture, fixed, and analyzed as described under "Experimental Procedures."

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Distribution and colocalization of GFP-4 deletion constructs with synaptobrevin II in cultured hippocampal neurons. a, d, and g, fluorescence patterns of neurons from low density hippocampal cultures infected with GFP-4 deletion constructs (a, N terminus; d, C terminus; g, GFP-4(50-407)). b, e, and h, hippocampal cells were stained with an anti-synaptobrevin II antibody and visualized with an Alexa546-coupled secondary antibody. c, f, and i, overlays of a and b (N terminus), d and e (C terminus), and g and h (GFP-4(50-407)) demonstrate that GFP-4-N terminus and GFP-4-C terminus (domains I and V), but not GFP-4(50-407), are colocalized with the synaptic vesicle marker synaptobrevin II (yellow). Scale bars = 10 µm for a, b, d, e, g, and h and 2 µM for c, f, and i.

To investigate the physiological role of GFP-₄, we measured EPSCs in the whole cell, voltage clamp configuration from autaptic hippocampal neurons infected with GFP-₄ and GFP-₄(50-407) and compared them with the EPSCs from noninfected neurons. As shown in Fig. 5, c and d, GFP-₄(50-407) assembled to form a functional Ca²⁺-channel when expressed together with the _1A- and ₂-subunit in HEK293 cells. The channel revealed inactivation properties comparable with the GFP-₄ assembled channels and increased the whole cell peak current measured at +10 mV (_1A, 48 ± 4 pA (n = 5); _1A/₂/GFP-₄(50-407), 1715 ± 390 pA (n = 17)). Since GFP-₄(50-407) did not colocalize with synaptobrevin II, specific presynaptic effects on synaptic transmission should become obvious only in the presence of GFP-₄, but not in the presence of GFP-₄(50-407). Recordings were performed in the presence of 30 µM Cs²⁺, which reduces the EPSC amplitude by >90%,² to more easily detect changes in EPSC size and facilitation properties between infected and noninfected cells. EPSC amplitudes for infected cells were related to the average EPSC size measured for noninfected autaptic hippocampal neurons prepared and measured on the same day. The average EPSC size increased drastically from 100 ± 20% (n = 46) for noninfected neurons to 334 ± 63% (n = 40, p < 0.01) for GFP-₄-infected neurons, but decreased for GFP-₄(50-407) infected neurons to 50 ± 17% (n = 34) (Fig. 7, a and b), indicating an increased transmitter release in the presence of GFP-₄ but not GFP-₄(50-407).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effects of GFP-4 and GFP-4(50-407) on EPSC amplitude of autaptic hippocampal neurons. a, representative autaptic EPSC traces from noninfected neurons and infected GFP-4(50-407) and GFP-4 neurons. EPSCs were elicited by a 2-ms depolarizing pulse to +10 mV. b, mean autaptic EPSC amplitude at 0.2 Hz stimulation in isolated hippocampal neurons. Right, EPSCs of infected neurons were compared with EPSCs of noninfected hippocampal cells (control 1 and control 2) recorded from the same cell preparation. The average EPSC amplitude from GFP-4-infected neurons was significantly larger (p < 0.01; two-tailed t test) than noninfected neurons, whereas the average EPSC amplitude from GFP-4(50-407) infected neurons was reduced compared with noninfected neurons. However, the effect was not significant. Left, EPSCs were related to the EPSCs of noninfected hippocampal cells recorded on the same day. c, number of EPSC measurements in hippocampal cells in percent for noninfected (control) and infected GFP-4 and GFP-4(50-407) hippocampal neurons. Autaptic EPSCs recordings from hippocampal neurons were categorized in 3 groups: no EPSC could be detected, EPSC amplitudes smaller than 100 pA, and EPSC amplitudes larger than 100 pA. The diagram reveals that 58% (control 1) and 41% (control 2) of noninfected, 57% of GFP-4(50-407)-infected, and only 18% of GFP-4-infected hippocampal neurons have EPSCs smaller than 100 pA, whereas 54% of GFP-4-infected hippocampal neurons and only 23% (control 1) and 31% (control 2) of noninfected and 21% of GFP-4(50-407)-infected hippocampal neurons have EPSCs larger than 100 pA. Number of neurons examined: control 1 = 57; control 2 = 29; GFP-4 = 56; GFP-4(50-407) = 44.

Short term synaptic enhancements during repetitive synaptic activity are attributable to a presynaptic increase in the number of transmitter quanta released. Ca²⁺ entry during the conditioning stimulation is required for their induction (23, 24). We therefore analyzed the effects of GFP-₄ and GFP-₄(50-407) on the facilitation properties of autaptic hippocampal neurons for EPSCs, which were larger than 80 pA and of comparable size in the infected and noninfected neurons. The ratios between the amplitudes of the first evoked EPSCs and the fourth evoked EPSCs for low and high frequency stimulations were determined. Low frequency stimulation (0.2 Hz) produced no EPSC facilitation in control (0.83 ± 0.04 (n = 59)), GFP-₄-infected (0.86 ± 0.05 (n = 33)), or GFP-₄(50-407)-infected (0.92 ± 0.06 (n = 24)) cells (Fig. 8, a and b). In contrast, high frequency stimulation (20 Hz) led to paired pulse facilitation of EPSCs. More importantly, paired pulse facilitation is increased significantly from 1.47 ± 0.07 (n = 44) in control cells to 2.67 ± 0.29 (n = 39) in GFP-₄-infected cells (p < 0.01) but not in GFP-₄(50-407)-infected cells (1.17 ± 0.06 (n = 25)) (Fig. 8, c and d), indicating a presynaptic function of GFP-₄ on paired pulse facilitation.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effects of GFP-4 and GFP-4(50-407) on paired pulse facilitation of autaptic hippocampal neurons. a, representative autaptic EPSC traces from noninfected neurons (control) and neurons infected with GFP-4 or GFP-4(50-407), respectively. 30 EPSCs were elicited at 0.2 Hz stimulation; the first and fourth EPSCs of one representative experiment are shown. b, EPSC ratios for 0.2 Hz stimulation are not significantly different between hippocampal neurons (control, white bar) and neurons infected with GFP-4 (dark gray bar) or GFP-4(50-407) (light gray bar). c, representative autaptic EPSC traces from hippocampal neurons (control) and neurons infected with GFP-4 or GFP-4(50-407), respectively. 30 EPSCs were elicited at 20 Hz stimulation and the first and fourth EPSCs of one representative experiment are shown. d, EPSC ratios for 20 Hz stimulation are significantly different between noninfected neurons (control; white bar) or GFP-4(50-407) (light gray bar)- and GFP-4 (dark gray bar)-infected neurons (p < 0.01, two-tailed t test). EPSC ratios were calculated by dividing the amplitude of the first EPSC by the fourth EPSC within each set of experiment.

To investigate whether the gating properties of a specific Ca²⁺-channel type assembled with different -subunits may account for the altered facilitation properties observed with overexpressed ₄-subunits in cultured hippocampal neurons, we analyzed Ca²⁺-currents (I_Ca²⁺) during high frequency stimulation of P/Q-type channels assembled with ₄ and _1b. We transfected HEK293 cells with _1A/₂ and ₄ or _1b, respectively, and elicited I_Ca²⁺ by a 20 Hz stimulation protocol. P/Q-type channels assembled with the _1b-subunit inactivated faster (32.5 ± 2.1 ms (n = 21)) and to a higher degree (87 ± 2% (n = 17)) than channels assembled with the ₄-subunit (54.9 ± 7.5 ms (n = 31); 74 ± 2% (n = 25)) when 10-ms test pulses to +10 mV were applied (Fig. 9). 20 Hz application of 2-ms test pulses to +10 mV had no effect on the size of P/Q-type channel currents assembled with ₄, whereas I_Ca²⁺ through _1b assembled channels decreased slowly (249 ± 22 ms (n = 20) by 22.5 ± 1.8%) (Fig. 9). Thus, the assembly of P/Q-type Ca²⁺-channel with different -subunits at the presynapses may be responsible for the increased facilitation in the presence of overexpressed ₄.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Effects of high frequency stimulation on P/Q-type Ca2+-currents in HEK293 cells. a, representative ICa2+ traces from HEK293 cells transfected with 1A, 2, and 4 (left traces) and 1A, 2, and 1b (right traces). 20 tail currents were elicited by a 2-ms (upper traces) or 10-ms (lower traces) voltage pulse from 70 mV to +10 mV every 50 ms (20 Hz stimulation). b, inactivation time constants for P/Q-type channels assembled with 1b or 4 were determined by fitting the decreasing tail currents with a single exponential. Inactivation time constant for P/Q-type channels assembled with 4 for the 2-ms test pulse could not be determined (n.d.) c, a larger reduction in ICa2+ is evident with 1b- compared with 4-transfected cells following 2- and 10-ms test pulses. The percent current reduction was determined by comparing the size of the first elicited tail current and the last elicited tail current within the stimulation protocol.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we describe the synaptic distribution and function of the Ca²⁺-channel ₄-subunit in cultured hippocampal neurons. Overexpression of a functional GFP-₄ fusion protein in cultured hippocampal neurons revealed a punctate staining pattern similar to the distribution of synaptic proteins synapsin I, synaptophysin (25), Munc-13-1 (26), syntaphilin (18), and synaptobrevin (27, 28). Colocalization of GFP-₄ with synaptobrevin II, a highly enriched presynaptic vesicle protein involved in transmitter release (29), and the presynaptic P/Q-type Ca²⁺-channel _1A-subunit suggests that GFP-₄ is also targeted to the presynaptic terminals. The recent finding that _1A and synaptobrevin are colocalized in mobile transport packets in hippocampal neurons (30) supports our results. Targeting signals to specific organelles have been identified in the extracellular and transmembrane domains of various proteins (31, 32). Infection of cultured hippocampal neurons with viral vectors is useful for identifying specific axonal sorting signals. For example, synaptobrevin contains a 92-aa long N-terminal region that is responsible for axonal targeting (28), whereas the metabotropic glutamate receptor 7 contains a 60-aa long cytoplasmic domain that mediates both axonal and dendritic targeting (33). To detect sequences responsible for targeting the ₄-subunit to presynaptic sites, we made use of the functional map and domain assignments suggested by Walker and De Waard (21) and Hanlon et al. (22). The identity of the N and C termini domains is only 2-3%, and these domains may therefore encode for different targeting signals among the -subunits as suggested by the colocalization of the N and C termini GFP fusion constructs with synaptobrevin. In addition, a _1b-specific acidic sequence in the C terminus has been described as responsible for membrane association of the _1b-subunit (34). A comparison of the targeting sequences from the ₄-subunit and the presynaptically transported proteins synaptobrevin II and mGluR7 did not reveal any homology. Therefore, the N- and C-terminal targeting sequences of Ca²⁺-channel ₄-subunit are distinct from other known presynaptic targeting signals. Interestingly, the C terminus of ₄ has been identified as a specifc, low affinity interaction site with the C terminus of _1A (35). The specific interaction between _1A- and ₄-subunits and the targeting to the presynapse mediated by ₄ may indicate how P/Q-type Ca²⁺-channel complexes are assembled at the presynapse. However, the assembly of ₄-subunits with other ₁-subunits at the presynapse cannot be excluded.

Physiological studies point to a presynaptic function of the Ca²⁺-channel ₄-subunit. For example, in lethargic mice where no functional ₄-subunit is expressed (36), the excitatory synaptic transmission in the thalamus and hippocampus, as well as the KCl-induced Ca²⁺-uptake through P/Q-type channels in thalamic and neocortical synaptosomes, is reduced (37-39). In our study, we demonstrate a synaptic function of ₄-subunits in synaptic transmission because overexpression of GFP-₄ but not GFP-₄(50-407) increased EPSC amplitude and paired pulse facilitation. One of the factors contributing to facilitation and EPSC size is the amount of Ca²⁺-influx into the presynaptic terminal through voltage-gated Ca²⁺-channels. -subunits have been described as determining the transport of the pore-forming ₁-subunits to the cell surface and increasing the number of channels in the plasma membrane (3-7). An increase in the presynaptic Ca²⁺-channel concentration may account for the larger EPSC amplitudes due to a higher release probability. We therefore measured somatic non-L-type Ca²⁺-currents in GFP-₄ infected and noninfected hippocampal neurons and observed a slight but not significant increase in current amplitude for GFP-₄ infected cells (0.748 ± 0.255 nA (n = 9)) compared with noninfected neurons (0.446 ± 0.123 nA (n = 9)). Thus, other effects leading to an increase in EPSC amplitude and facilitation cannot be excluded. For example, if the Ca²⁺-channel concentration at autaptic synapses does not change and the release probability at a single synapse is not affected by overexpression of ₄, then the successful transmission for a given action potential would remain unchanged. The formation of new synaptic contacts, new active sites, or postsynaptic effects, such as an increase in ligand gated cation channels, would explain our results. However, the altered facilitation properties point to a presynaptic mechanism.

The increase in the paired pulse facilitation ratio may reflect the slow inactivation time of presynaptic Ca²⁺-channels assembled with the ₄-subunit for a limited access of Ca²⁺, because ₄ causes Ca²⁺-channels to inactivate slower compared with channel complexes containing _1b or ₃ (19, 40). This hypothesis is underlined by our results showing that in the presence of ₄-subunits, inactivation is slower and current reduction is decreased compared with P/Q-type channels expressed with _1b. Therefore, Ca²⁺-channel complexes containing ₄-subunits at the presynapse may contribute to increased synaptic facilitation.

In summary, our findings suggest a presynaptic function of Ca²⁺-channel ₄-subunits in synaptic transmission. The specific targeting of ₄ depends on its nonconserved N and C termini, which is critical for the localization of the subunit to synaptic terminals and for the short term modulation of synaptic transmission.

	ACKNOWLEDGEMENTS

We thank B. Rudo, A. Bührmann, and I. Herfort for excellent technical assistance. We are grateful to Drs. T. P. Snutch and E. Perez-Reyes for cDNAs, Dr. R. Jahn for the monoclonal antibody to synaptobrevin II, Dr. C. Rosenmund for helpful comments, and Drs. J. P. Ruppersberg and E. Neher for generous support.

	FOOTNOTES

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft (He2471/5-1 to S. H. and Re1092/3-2 to J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 049-7071-2978279; Fax: 049-7071-87815; E-mail: stefan.herlitze@uni- tuebingen.de.

Published, JBC Papers in Press, August 7, 2000, DOI 10.1074/jbc.M004653200

	ABBREVIATIONS

The abbreviations used are: GFP, green fluorescent protein; aa, amino acid(s); EPSC, excitatory post-synaptic current; HEK, human embryonic kidney.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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