Kittler et al. 10.1073/pnas.0506653102. |
Table 1. Analysis of GABAA receptor–P2 interactions by using Surface Plasmon Resonance
Peptide | Binding to native AP2 | ||
K a, M–1 ´ s–1 | K d, s–1 | K d, nM | |
CKTHLRRRSSQLK | 9.3 ´ 103 | 2.8 ´ 10–3 | 300 |
CKTHLRRRS*S*QLK | 2.1 ´ 103 | 3.9 ´ 10–3 | 1900 |
*, phosphorylated. SPR binding data of the nonphosphorylated (pepb3: CKTLRRRSSQLK) and phosphorylated (pepb3-phos: CKTLRRRS*S*QLK) peptides to purified adaptor protein (AP2) from the brain.
Supporting Materials and Methods
Affinity Purification Assays.
GST fusion protein affinity chromatography (GST pull down assays) were carried out in Hepes buffer: 25 mM Hepes, pH 7.6/150 mM NaCl/5 mM EDTA/1% Triton-X-100 and a mixture of protease inhibitors as described in ref. 1. In vitro translation of adaptor protein 2 (AP2) subunits was carried out by using the Quick coupled transcription/translation system (Promega) according to the manufacturer’s instructions as described in ref. 2. For pull downs from brain lysate, solubilized brain lysate was produced by homogenizing rat brain in Hepes buffer followed by centrifugation at 50,000 ´ g for 1 h and used in pull down assays as described in ref. 1. For assays with phosphorylated fusion proteins, GST fusion proteins encoding the intracellular domain (ICD) of GABAA receptor (GABAAR) b3 subunit were phosphorylated in vitro by purified PKC (provided by A. Nairn, The Rockefeller University, New York) or protein kinase A (PKA) (Promega) as described in ref. 3. For mock phosphorylation, the identical in vitro phosphorylation reaction was carried out in the absence of an added kinase. These phosphorylated or mock phosphorylated fusion proteins were then attached to glutathione agarose beads and used in pull down assays with 35S-labeled m2 as described above.Surface Plasmon Resonance (SPR).
Binding of AP2 and m2 was recorded in real time by using a surface plasmon-based biosensor (BIAcore 3000, Biacore). AP2 was purified from a pig brain as described in ref. 4 and soluble m2 and the m2 mutant W421A mutant were purified as histidine-tagged fusion proteins according to a standard protocol (Qiagen) from the E. coli strain DH-5a. For SPR, the synthetic peptides pepb3 (CKTHLRRRSSQLK) and pepb3-phos (CKTHLRRRS*S*QLK) were immobilized via their amino-terminal cysteine by using the thiol coupling method according to the instructions of the manufacturer (Bia) on a CM5 sensor surface. Immobilization resulted in a stable positive shift of the baseline by 1,000 resonance units. After coupling, the surface was washed by three consecutive injections of NaOH and with running buffer (20 mM Hepes, pH 7.4/2 mM DTT/300 mM NaCl). Binding of AP2 and m2 was recorded at a flow rate of 20 ml/min for 2 min (association phase), followed by washing with running buffer (dissociation phase) for 2 min and regeneration of the surface by a pulse injection (5 sec) with 50 mM NaOH/0.5% SDS. AP2 was used at four concentrations ranging from 250 nM to 1 mM, and m2 was used from 500 nM to 5 mM. All proteins passed over the sensor surface were centrifuged for 15 min at 150,000 ´ g before any SPR measurements to remove aggregates. The rate constants were calculated with the evaluation software (Biacore) assuming a 1:1 mode of interaction.Acute-Dissociation Procedure and Neuronal Culture.
Cortical neurons from young adult (3-5 weeks postnatal) rats were acutely dissociated by using procedures similar to those described in ref. 5. All experiments were carried out with the approval of State University of New York at Buffalo Animal Care Committee. After incubation of brain slices in NaHCO3-buffered saline, frontal cortex was dissected and placed in an oxygenated chamber containing protease type XIV (1.2 mg/ml) (Sigma) in Hepes-buffered Hank’s balanced salt solution (Sigma) at room temperature. After 30 min of enzyme digestion, the tissue was rinsed three times in the low Ca2+, Hepes-buffered saline and mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35-mm Lux Petri dish that was then placed on the stage of a Nikon inverted microscope.Whole-Cell Recordings.
Whole-cell recordings of currents in isolated neurons used standard voltage-clamp techniques (5, 6). The internal solution consisted of 180 mM N-methyl-glucamine (NMG)/40 mM Hepes/4 mM MgCl2/0.5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate/12 mM phosphocreatine/2 mM Na2-ATP/0.2 mM Na3GTP/0.1 mM leupeptin, pH 7.2-7.3/265-270 mOsm/liter. The external solution consisted of (in mM): 135 mM NaCl/20 mM CsCl/1 mM MgCl2/10 mM Hepes/5 mM BaCl2/10 mM glucose/0.001 mM TTX, pH = 7.3-7.4/300-305 mOsm/liter. Recordings were obtained with an Axon Instruments 200B patch clamp amplifier that was controlled and monitored with an IBM PC running pclamp (v.8) with a DigiData 1320 series interface (Axon instruments, Foster City, CA). Electrode resistances were typically 2–4 MW in the bath. After seal rupture, series resistance (4–10 MW) was compensated (70–90%) and periodically monitored. The cell membrane potential was held at 0 mV. GABA (50 mM) was applied for 2 sec every 30 sec. Drugs were applied with a gravity-fed "sewer-pipe" system. The array of application capillaries (»150 mM i.d.) was positioned a few hundred microns from the cell under study. Solution changes were effected by the SF-77B fast-step solution stimulus delivery device (Warner Instruments, Hamden, CT). The whole-cell patch technique (5, 6) was used for recordings of miniature inhibitory synaptic currents (mIPSCs) in cultured neurons. Electrodes were filled with the following internal solution: 100 mM CsCl/30 mM NMG/10 mM Hepes/1 MgCl2/4 mM NaCl/5 mM EGTA/0.8 mM QX314/12 mM phosphocreatine/2 mM MgATP/0.2 mM Na3GTP/0.1 mM leupeptin, pH = 7.2-7.3/265-270 mOsm/liter. The cultured neurons were submerged in continuously flowing oxygenated artificial cerebrospinal fluid containing Tetrodotoxin (0.5 mM). For blocking glutamate transmission, the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 mM) and NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (25 mM) were added to the recording solution. The cell membrane potential was held at –60mV. Data analyses were performed with axograph (Axon Instruments, Union City, CA), kaleidagraph (Albeck Software, Reading, PA) and mini analysis program (Synaptospft, Leonia, NJ).1. Kittler, J. T., Delmas, P., Jovanovic, J. N., Brown, D. A., Smart, T. G. & Moss, S.J. (2000) J. Neurosci. 20, 7972–7977.
2. Haucke, V., Wenk, M. R., Chapman, E. R., Farsad, K. & De Camilli, P. (2000) EMBO J. 19, 6011–6019.
3. Jovanovic, J. N., Thomas, P., Kittler, J. T., Smart, T. G. & Moss, S. J. (2004) J. Neurosci. 24, 522–530.
4. Fingerhut, A., von Figura, K. & Honing, S. (2001) J. Biol. Chem. 276, 5476–5482.
5. Feng, J., Cai, X., Zhao, J. & Yan, Z. (2001) J. Neurosci. 21, 6502–6511.
6. Chen, G., Greengard, P. & Yan, Z. (2004) Proc. Natl. Acad. Sci. USA 101, 2596–2600.