in the presence and absence of 300mM extracellular Ba2þ. Current–voltage relations fortransfected and control neurons were calculated by recording whole-cell currents undervoltage clamp. Voltage steps in 10-mV increments were applied every 1 s. Evoked synapticcurrents were recorded using pipettes whose internal solution was supplemented withQX-314 (4 mM), and in the presence of 50–100mM picrotoxin in the extracellular mediumto isolate excitatory synapses. Brief 1-ms pulses were applied to the entire field of neuronsusing platinum wires separated by about 5 mm. The applied voltage was systematicallyvaried for each recording until the response amplitude became saturated, indicatingreliable activation of all axons innervating the recorded neuron. Responses were measuredat a holding potential of 270 mV. To record action-potential firing, without perturbingthe intracellular environment, we used the cell-attached patch-clamp method. Pipettessimilar to those for whole-cell recordings were used, and tight seals were obtained withoutbreak-in. Current recordings (under voltage clamp at 270 mV) allowed unambiguousdiscrimination of spikes with high signal-to-noise ratios.FM4-64 imagingTo identify functional presynaptic terminals, we labelled recycling synaptic vesicles using10mM FM4-64 (Molecular Probes). Neurons were depolarized for 60 s usinghyperkalaemic solution (in mM): 78.5 NaCl, 60 KCl, 10 HEPES, 10D-glucose, 2 CaCl2, 1.3MgCl2, 0.05 AP5 (D(-)-2-amino-5-phosphonovaleric acid), 0.005 CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), and 0.001 TTX. Coverslips were then washed in regularextracellular medium without FM4-64 for 10 min before imaging to reduce thebackground fluorescence caused by non-internalized dye binding to the cell membrane.This protocol has been shown to provide an estimate of the total recycling pool of vesicles.Cells were then imaged in dye-free buffer containing blockers. Earlier experiments alsoincluded a de-staining step to release dye from vesicles, and images after de-staining weresubtracted from the initial image. As the initial fluorescence and releasable fluorescencewere strongly correlated, in most experiments we skipped the de-staining step. Imagestacks (Z steps of 1mm, 7–10 steps) were obtained using a confocal microscope (OlympusFluoview attached to a BX50WI, £ 40, 0.8NA water lens). EGFP and FM4-64 signals wereacquired simultaneously using 488-nm excitation, and 510–550-nm band pass and 585long-pass emission filters, respectively. Transmitted light images were taken separately toidentify cell bodies and processes of non-transfected neurons. We chose to measure thedensity of presynaptic terminals in the proximal regions of the dendrites (about 100mmfrom the soma) as these could be identified unequivocally by the EGFP fluorescence.Image analysis was performed in a blind manner with respect to Kir2.1 and mutKir2.1neurons. 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