Fig. 4

Kinetics of the Cl⁻ extrusion in KCC2a-, KCC2b-, and mock- transfected Neuro-2a cells. Recordings of [Cl⁻]_i changes (Δ[Cl⁻]_i) in Neuro-2a cells transfected with KCC2a (a), KCC2b (b), and empty vector (c) constructs. Left panels show individual recordings of representative cells for each of the constructs; right panels demonstrate quantifications of the individual recordings. After initial baseline acquisition (4 min), cells were loaded with chloride by applying 100 µM glycine in 50 mM K⁺ ECS (10 min). Washing cells with normal K⁺ ECS for 5–10 min was accompanied by rapid decrease in [Cl⁻]_i. The rate of the chloride extrusion Δ[Cl⁻]_i/Δt was much faster in cells transfected with KCC2a (a) and KCC2b (b) constructs compared to the mock-transfected cells (c). In the second phase of the experiment, the whole procedure was repeated in the presence of 500 µM furosemide, a known inhibitor of K–Cl cotransport. The initial rate of [Cl⁻]_i reduction (mM/min) was calculated using the first half of each slope. For each individual cell, the rate of furosemide-sensitive Cl extrusion was calculated as a difference between the corresponding rates of [Cl⁻]_i extrusion in the presence and absence of furosemide