Figure 1
CKII activity is necessary for ENaC trafficking to the plasma membrane and channel activity. (A) Fluorescence micrographs of COS-7 cells expressing eYFP-ENaC in the absence (top) and presence (bottom) of TBB treatment prior to photobleaching (left) and 10 s (middle) and 10 min (right) after photobleaching. Cells were treated with 200 nM TBB for 30 min. Images were collected with TIRF microscopy. (B) Time course of relative FRAP at the plasma membrane for cells expressing eYFP-ENaC in the absence (black circles) and presence of TBB (black squares). Summary data from experiments (n = 8–10 cells from 2–3 distinct transfections) identical to those shown in (A). (C) Summary graph of relative FRAP 10 min after photobleaching in cells expressing eYFP-ENaC in the absence (black circles, gray bar) and presence (black squares, white bar) of TBB. Summary data from experiments identical to those shown in (A). *P < 0.05 vs. absence of TBB. (D) Overlays of typical macroscopic current traces from representative CHO cells expressing mENaC in the absence (top) and presence of TBB (bottom) before and after 10 µM amiloride (dotted lines). Cells were treated with 200 nM TBB for 30 min prior to the recording. Currents elicited by voltage ramps stepped from a holding potential of 40 mV to 60 mV and ramped to − 100 mV. (E) Summary graph of ENaC activity (amiloride-sensitive current density at -100 mV) quantified in whole-cell voltage clamped CHO cells transfected with mENaC in the absence (black circles, gray bar) and presence (black squares, white bar) of TBB. Summary data from experiments (n = 8–9 cells from 3–5 distinct transfections) identical to those shown in (D). *P < 0.05 vs. absence of TBB.
