guanosine-triphosphate and Cystic-Fibrosis

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl- conductance (CFTR G(Cl)) in the native sweat duct (SD). We permeabilized the basolateral membrane with alpha-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G(Cl) activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-gamma-S (100 microm) also activates CFTR G(Cl) in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-gamma-S increased CFTR G(Cl) by 44 +/- 20 mS/cm(2) (mean +/- se; n = 7). GDP (10 mm) inhibited G-protein activation of CFTR G(Cl) even in the presence of GTP-gamma-S. The heterotrimeric G-protein activator (AlF(4-) in the cytoplasmic bath activated CFTR G(Cl) (increased by 51.5 +/- 9.4 mS/cm(2) in the presence of 5 mm ATP without cAMP, n = 6), the magnitude of which was similar to that induced by GTP-gamma-S. Employing immunocytochemical-labeling techniques, we localized Galphas, Galphai, Galphaq, and Gbeta at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G(Cl) in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G(Cl) activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G(Cl) activity.

Topics: Adenosine Triphosphate; Adult; Aluminum Compounds; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Humans; Immunohistochemistry; In Vitro Techniques; Male; Mutation; Sodium Channels; Sodium-Potassium-Exchanging ATPase; Sweat Glands

In a previous study [26] we described the properties of potassium channels in cultured respiratory cells derived from cystic fibrosis patients (CF) and normal individuals (N). In the present study we examine the regulatory mechanisms of these channels by the patch clamp technique. Since there were no apparent differences in the properties of CF and N K+ channels the results were pooled. In the excised inside/out configuration the channel was blocked by different K+ channel blockers. Barium (5.10(-3) mol/l), tetraethylammoniumchloride (5.10(-3) mol/l), quinidine (10(-3) mol/l) and lidocaine (5.10(-3) mol/l), when added to the cytosolic side, inhibited K+ channels reversibly. An increase in the calcium concentration from 10(-7) mol/l to 10(-6) mol/l led to a marked increase in the open channel probability (Po). Further increases in Ca2+ concentration increased Po only slightly. No pH effects on the cytosolic side of the channel were observed. The channel open probability was reduced when ATP was present on the cytosolic side at a concentration of 10(-4) mol/l to 10(-3) mol/l. Non hydrolysable adenosine 5'-[beta, gamma-methylene] triphosphate had the same inhibitory effect as ATP. The inhibition by ATP was blunted by the simultaneous addition of 1 mmol/l ADP. The inhibition of K+ channels by cytosolic ATP may represent a channel regulatory mechanism in the intact cell. This would allow for coupling between the activity of the (Na+ + K+)-pump and the basolateral K+ conductance.

Topics: Adenosine Triphosphate; Barium; Calcium; Cell Membrane; Cell Membrane Permeability; Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Epithelium; Guanosine Triphosphate; Humans; Hydrogen-Ion Concentration; Lidocaine; Membrane Potentials; Nasal Polyps; Potassium; Potassium Channels; Quinidine; Sodium; Tetraethylammonium; Tetraethylammonium Compounds