Cytosolic potassium controls CFTR deactivation in human sweat duct

Absorptive epithelial cells must admit large quantities of salt (NaCl) during the transport process. How these cells avoid swelling to protect functional integrity in the face of massive salt influx is a fundamental, unresolved problem. A special preparation of the human sweat duct provides critical insights into this crucial issue. We now show that negative feedback control of apical salt influx by regulating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel activity is key to this protection. As part of this control process, we report a new physiological role of K+ in intracellular signaling and provide the first direct evidence of acute in vivo regulation of CFTR dephosphorylation activity. We show that cytosolic K+ concentration ([K+]c) declines as a function of increasing cellular NaCl content at the onset of absorptive activity. Declining [K+]c cause parallel deactivation of CFTR by dephosphorylation, thereby limiting apical influx of Cl− (and its co-ion Na+) until [K+]c is stabilized. We surmise that [K+]c stabilizes when Na+ influx decreases to a level equal to its efflux through the basolateral Na+-K+ pump thereby preventing disruptive changes in cell volume.

Bumetanide blocks CFTRG Cl in the native sweat duct

Bumetanide is well known for its ability to inhibit the nonconductive Na+-K+-2Cl−cotransporter. We were surprised in preliminary studies to find that bumetanide in the contraluminal bath also inhibited NaCl absorption in the human sweat duct, which is apparently poor in cotransporter activity. Inhibition was accompanied by a marked decrease in the transepithelial electrical conductance. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is richly expressed in the sweat duct, we asked whether bumetanide acts by blocking this anion channel. We found that bumetanide 1) significantly increased whole cell input impedance, 2) hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membrane potential, 4) increased the ratio of apical-to-basolateral membrane resistance, and 5) decreased transepithelial Cl− conductance ( G Cl). These results indicate that bumetanide inhibits CFTR G Clin both cell membranes of this epithelium. We excluded bumetanide interference with the protein kinase A phosphorylation activation process by “irreversibly” phosphorylating CFTR [by using adenosine 5′- O-(3-thiotriphosphate) in the presence of a phosphatase inhibition cocktail] before bumetanide application. We then activated CFTR G Clby adding 5 mM ATP. Bumetanide in the cytoplasmic bath (10−3 M) inhibited ∼71% of this ATP-activated CFTR G Cl, indicating possible direct inhibition of CFTR G Cl. We conclude that bumetanide inhibits CFTR G Clin apical and basolateral membranes independent of phosphorylation. The results also suggest that >10−5 M bumetanide cannot be used to specifically block the Na+-K+-2Cl−cotransporter.

Intracellular Cl activity: evidence of dual mechanisms of cl absorption in sweat duct

The human sweat duct (SD) reabsorbs NaCl from lumen to blood over a wide range of luminal concentrations. The physiological strategies employed by the SD to cope with such extreme transport loads remain elusive. When we employed intracellular Cl-sensitive microelectrodes, we found that at high (150 mM) luminal NaCl concentrations ([NaCl]) transcellular Cl absorption occurs through passive diffusion that is evidenced by a large Cl conductance (GCl) in both cell membranes and by a favorable electrochemical driving force for Cl (delta psi Cl) across the apical and basolateral membranes. However, lowering the luminal [NaCl] to 15 mM markedly altered the electrochemical gradient for Cl and reversed the direction of delta psi Cl. Under these conditions, passive absorption of Cl was not feasible, so that Cl can only be absorbed by a nonconductive transport carrier. We surmise that, in the face of such changes in delta psi Cl as a function of luminal [NaCl], continuous transcellular Cl transport in SD could only be sustained if both electroconductive and carrier-mediated Cl transport are present in the SD.