Patch Clamp Electrophysiology.

HEK293 cells were plated on glass coverslips in dishes and incubated for 20 to 44 h before calcium phosphate transfection with 3 µg cDNA. For macroscopic current recordings, the cDNA transfection was done with the Rat GluA2 (Q) pRK5 vector encoding enhanced green fluorescent protein after an internal ribosome entry site. Single-channel recordings from outside-out patches were performed 24 h after transfection. For single-channel recordings, we used a plasmid ratio approach to obtain sparse expression (67), whereby the rat GluA2 (Q) vector was cotransfected with enhanced green fluorescent protein and empty vector (pCDNA3.1+) in a ratio of 1:63:313. Standard extracellular solution contained 150 mM NaCl, 0.1 mM MgCl₂, 0.1 mM CaCl₂, 5 mM HEPES, and 10 µM EDTA and was titrated with NaOH to a pH of 7.3. We included EDTA to chelate trace contamination by divalent ions. The internal solution contained 115 mM NaCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 10 mM NaF, 5 mM Na₄BAPTA, 10 mM Na₂ATP, and 5 mM HEPES, titrated to a pH of 7.3 with NaOH. Pipettes were mounted in an ISO holder (G23 Instruments) and had a resistance of 3 MΩ for macroscopic current recordings. For single-channel recording, pipettes were fire polished to a resistance of 10 to 25 MΩ and were coated with Sylgard. The junction potential between the pipette and Na⁺-bath solution (E_bath-E_pip, considering Na⁺, Cl⁻, and F⁻ mobilities) was 3.7 mV (68). For reversal potential experiments with Cs, we substituted NaCl in the extracellular solution with CsCl and titrated the pH to 7.3 with CsOH. For single-channel recordings in near-symmetrical K⁺ or Cs⁺, in external solutions, we substituted NaCl with KCl or CsCl, respectively, and titrated to pH 7.3 with KOH or CsOH, respectively. The Cs internal solution contained 115 mM CsCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 10 mM CsF, 5 mM Cs₄BAPTA, 10 mM K₂ATP, and 5 mM HEPES, titrated to a pH of 7.3 with CsOH. The potassium internal solution contained 115 mM KCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 10 mM CsF, 5 mM Cs₄BAPTA, 10 mM K₂ATP, and 5 mM HEPES, titrated to a pH of 7.3 with KOH. Cyclothiazide (Hello Bio) was prepared as a 100-mM stock solution in DMSO and used at 100 µM (giving a final concentration of 0.1% DMSO). EDTA stock solution was prepared in NaOH. Reagents were obtained from Carl Roth GmbH, Sigma Aldrich, or Toronto Research Chemicals, unless otherwise noted. For fast perfusion of outside-out patches, the perfusion tools were made with custom-designed four-barrel square profile glass (Vitrocom) mounted on a piezo electric stack (Physik Instrument). Currents were filtered at 10 kHz (−3 dB cutoff, 8-pole Bessel) with an Axopatch 200B amplifier (Molecular Devices). For analog–digital conversion, an InstruTECH ITC-18 digitizer (HEKA Elektronik Dr. Schulze GmbH) was used at 40-kHz sampling rate. Data were recorded and analyzed with AxoGraph X (AxoGraph Scientific).

For single-channel conductance measurements, in each record, we ran a ramp protocol (−120 to +120 mV, 1.2 V ⋅ s⁻¹) both before and during glutamate application (10 mM) to the outside-out patch. The leak current recorded during the no-glutamate ramp was subtracted from the current recorded during glutamate application. Stretches of the recording corresponding to one open channel were selected and the open levels were fit with a linear relation to obtain the chord conductance.

For macroscopic measurements of reversal potential and conductance, we alternated washing each patch with Cs⁺ and Na⁺ external solution. Slope conductances were fit to the traces over the 30-mV ranges at the extreme positive and negative ends of the ramp. For the slope conductance, we compared the conductance ratios (Cs versus Na inward and Na versus Na outward) from the same patch against the null (ratio = 1) using a paired t test. We assumed that there was a similar junction potential (within 0.5 mV) in both cases because the junction potential from the pipette to the Cs⁺ solution (−1.7 mV) is cancelled by a second junction potential from Cs⁺ solution back to the bath electrode in Na⁺ (4.9 mV) (69). We added this junction potential to the measured mean reversal potential. The flow rate through the local perfusion tool was low (<200 µL/min), meaning the overall bath Cs⁺ concentration remained low.

We used the GHK equation (70) to calculate permeability ratios based on shifts in reversal potentials:

where X was Na⁺ or Cs⁺.

Likewise, to calculate the putative shift in E_rev because of a minor chloride permeability, we took internal [X]_i and external [X]_O ion concentrations, where X was Na⁺, Cs⁺, or Cl⁻ permeability ratios of P_Cs:Na of 1.16:1, as measured, and assumed P_Cl:Na of either 1:220 (an upper estimate based on the observation of a single anion permeation in our potassium simulations) or zero (no chloride permeability). Cesium terms were only used for the bi-ionic condition of [Na⁺]_i to [Cs+]_o:

Statistical analysis and data plotting was done in IGOR Pro (Wavemetrics).