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Fig. 1. Kir2.1 channels are neither newly synthesized nor transported to the plasma membrane during the first 6 hours of differentiation. (A) Typical Kir2.1 current recorded from one myoblast cultured for 6 hours in differentiation medium (DM). Ba²⁺ (500 µM), as expected, inhibited the current. Voltage-steps were to -40, -60, -80, -100, -120 and -140 mV from a holding potential at -60 mV. Current-to-voltage relationships are shown in control condition and in the presence of 500 µM Ba²⁺.(B) Myoblasts were cultured in DM for 6 hours with or without 3 µg/ml cycloheximide (CHX). Kir2.1 current amplitude was assessed during a 300 ms step to -120 mV from a holding potential at -60 mV. The upper histogram represents the fraction of myoblasts expressing Kir2.1 current (5 pA) and the lower histogram the current density of the total population of myoblasts (including myoblasts with no current, i.e. <5 pA) in control and in cycloheximide conditions. Both histograms are from the same recordings. Leak currents were estimated either by linear extrapolation from the currents recorded at -40 and -60 mV or by addition of 500 µM Ba²⁺ to the external bath solution, and subtracted to the total current. Twenty-eight myoblasts (out of three different clones) with a mean cell capacitances of 32±3 pF were recorded under control conditions, and 46 myoblasts (out of three different clones) with a mean cell capacitances of 27±2 pF in the presence of cycloheximide. Asterisk indicates P<0.05 (and in subsequent figures). [³⁵S]-methionine incorporation was performed in the absence or presence of cycloheximide 3 µg/ml during 3 hours to evaluate protein synthesis. (C) Myoblasts were cultured in DM for 6 hours with or without 10 µg/ml Brefeldin A (BFA). Histograms represent the fraction of myoblasts with Kir2.1 current and the Kir2.1 current density as in B. Both histograms are from the same recordings. Nineteen myoblasts (out of four different clones) with a mean cell capacitances of 34±3 pF were recorded in control conditions, and 46 myoblasts (out of three different clones) with a mean cell capacitances of 23±2 pF in the presence of Brefeldin A. Representative pictures (confocal microscopy) of myoblasts transfected with Kir2.1-GFP and incubated immediately after transfection in the presence or absence of 10 µg/ml Brefeldin A for 12 hours. In the presence of Brefeldin A, Kir2.1-GFP channels do not reach the plasma membrane.

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