Figure 3. Repaglinide binds to DREAM and blocks DREAM activity on potassium channel gating.

(A) Affinity pull-down assay of recombinant GST, GST-DREAM, or GST-neurocalcin (2% input in lanes 1–3). For each recombinant protein, the binding signal is shown to uncoupled Sepharose (lane 4), or to Sepharose coupled to (i) repaglinide with 4 mM EGTA (lane 5) or with 4 mM Ca²⁺ (lane 6) and (ii) other benzoic acid derivatives (lanes 7–9; cyanic acid, aspirin, and 3,4,5-trimethyoxybenzoic acid, respectively). A representative blot is shown. (B) Surface plasmon resonance analysis of the binding of repaglinide (RP), CL-888, and glibenclamide (Glib) (all at 2 μM) to immobilized GST-DREAM. A representative curve is shown. (C and D) Effects of repaglinide, CL-888, or glibenclamide on Kv4.3 channel gating. (C) Concentration curve for the inhibitory effect of repaglinide or CL-888. Experiments in CHO cells transfected with Kv4.3 and DREAM. Blockade was measured as the reduction in the amount of charge crossing the cell membrane (estimated from the integral of the current signal) during the 250 ms pulses to +60 mV. Lines represent the fit of the data to a Hill equation. Each point represents mean ± SEM of 3 to 9 experiments. (D) Representative Kv4.3+DREAM currents recorded on transiently transfected CHO cells. Current traces were obtained after depolarization to +60 mV from a holding potential of 80 mV. Currents were recorded in control conditions and after perfusion with 5 μM glibenclamide (left), 100 nM CL-888 (center), or 100 nM repaglinide (right).