Cystic Fibrosis Transmembrane Conductance Regulator Inverts Protein Kinase A-mediated Regulation of Epithelial Sodium Channel Single Channel Kinetics

Abstract

Abnormal regulation of ion channels by members of the ABC transport protein superfamily has been implicated in hyperinsulinemic hypoglycemia and in excessive Na؉ absorption by airway epithelia in cystic fibrosis (CF). How ABC proteins regulate ion conductances is unknown, but must generally involve either the number or activity of specific ion channels. Here we report that the cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in CF, reverses the regulation of the activity of single epithelial sodium channels (ENaC) by cAMP. ENaC expressed alone in fibroblasts responded to activation of cAMP-dependent protein kinase with increased open probability (P o ) and mean open time, whereas ENaC co-expressed with CFTR exhibited decreased P o and mean open time under conditions optimal for PKA-mediated protein phosphorylation. Thus, CFTR regulates ENaC at the level of single channel gating, by switching the response of single channel P o to cAMP from an increase to a decrease.Recent studies (1, 2) have identified ENaC as the channel that mediates amiloride sensitive Na ϩ absorption in mammalian airways. In cystic fibrosis (CF), 1 ENaC-mediated Na ϩ absorption is increased 200 -300% in airway epithelia and, abnormally, further stimulated by raising intracellular cAMP (3). Because most CF mutations result in little if any functional CFTR in the apical cell membrane of affected epithelia (4), we inferred that normal CFTR must either down-regulate the number of active Na ϩ channels or decrease the activity of individual Na ϩ channels. In the present study we have studied the effects of cAMP-dependent protein-phosphorylating conditions on the single channel kinetics of ENaC expressed alone or together with CFTR in NIH 3T3 fibroblasts. EXPERIMENTAL PROCEDURES␣-, ␤-, and ␥-ENaC subunits were stably expressed in NIH 3T3 cell lines that had been previously transduced with a truncated (inactive) interleukin-2 receptor (ENaC alone cells) or with human CFTR (ENaC ϩ CFTR cells) (5). ENaC-mediated single channel currents were recorded from cell attached and excised membrane patches as described in the figure legends. RESULTSThe single channel conductance (4 -5 picosiemens) of ENaC expressed in NIH 3T3 fibroblasts, as well as cation selectivityand the slow gating pattern (MOT Ϸ 1 s), are similar to what has been reported for the cloned channel expressed in oocytes (6, 7) and for endogenously expressed ENaC in rat cortical collecting tubule (8) or A6 cells (9) (Fig. 1). These similar results in very different cells suggest that cell specific cytoskeletal or other elements are not critical determinants of the basic biophysical characteristics of ENaC. The basal conductance and amiloride sensitivity of ENaC were not affected by co-expression with CFTR ( Fig. 1).ENaC present in excised membrane patches exhibited a variable degree of rundown following excision. Rundown was partially reversed (Fig. 2A, panel i) or prevented ( Fig. 2A, panel ii) by exposure of the cytoplasmic surface to PKA catalytic subunit and 2 ...

Regulation of the Epithelial Na+ Channel by Cytosolic ATP

“…When NP o value was normalized to a value of 1 at Ϫ24 mV, the relative NP o was 2.54 Ϯ 0.68 (n ϭ 6) (p Ͻ 0.01) at Ϫ84 mV. Together, these results extend previous electrophysiological characterizations of rENaC stably expressed in 3T3 cells (10,11) and show that the biophysical properties of the macroscopic and microscopic currents attributable to the activity of rENaC expressed in these cells were similar to those observed in native Na ϩ -transporting epithelia, such as rat cortical collecting tubule (1) and other heterologous expression systems such as Xenopus oocytes and MDCK cells (3,16,30).…”

“…Cells-A previous study has shown that ␣␤␥rENaC-expressing 3T3 cells, but not untransfected 3T3 cells, exhibit an amiloride-sensitive conductance (10,11). By using the conventional whole-cell configuration with the standard cesium glutamate-rich pipette solution containing 2 mM ATP and lithium glutamate-rich bath solution, we first characterized in detail amiloride-sensitive macroscopic currents mediated by rENaC in this heterologous expression system.…”

Genistein Restores Functional Interactions between ΔF508-CFTR and ENaC in Xenopus Oocytes

“…Functional expression of WT-CFTR, but not ⌬F508-CFTR, is associated with an inhibition of func- tional ENaC expression in oocytes (31). Such CFTR-mediated inhibition of ENaC is associated with a decrease in Na ϩ channel open probability, not with changes in levels of ENaC mRNA or protein expression (8,42,43). The forskolin/IBMX-regulated inhibition of ENaC by CFTR does not require expression of the full-length CFTR protein.…”

Regulation of the Epithelial Sodium Channel by Serine Proteases in Human Airways

“…The physiologic importance of the relationship between ENaC activity in airways and ASL volume is highlighted by two disease states, cystic fibrosis (CF) and pseudohypoaldosteronism type 1. In CF, the loss of functional cystic fibrosis transmembrane conductance regulator (CFTR) protein results in excessive ENaC activity (3,4), sodium hyperabsorption (5), and ASL volume depletion (6). The depletion of ASL volume, in turn, causes the overlying mucus gel to collapse onto cilia and the cell surface, thus disrupting mucociliary clearance (7).…”

“…Similarly, while signals that raise intracellular cAMP in the kidney (e.g. vasopressin) increase ENaC currents by stimulating channel insertion into the plasma membrane (10,11), this second messenger pathway has little effect on amiloride-sensitive currents in normal airways (4). Finally, while patients with Liddle's syndrome manifest renal sodium hyperabsorption due to ENaC mutations (12)(13)(14), they do not appear to have sodium hyperabsorption across airway epithelia (15).…”