Search

Previous studies have characterized interactions between the ubiquitin ligase Nedd4-1 and the epithelial Na(+) channel (ENaC). Such interactions control the channel cell surface expression and activity. Recently, evidence has been provided that a related protein, termed Nedd4-2, is likely to be the true physiological regulator of the channel. Unlike Nedd4-1, Nedd4-2 also interacts with the aldosterone-induced channel activating kinase sgk-1. The current study uses surface plasmon resonance to quantify the binding of the four WW domains of Nedd4-2 to synthetic peptides corresponding to the PY motifs of ENaC and sgk-1. The measurements demonstrate that WW3 and WW4 are the only Nedd4-2 domains interacting with both ENaC and sgk-1 and that their binding constants are in the 1-6 microM range.

Epithelial Na+ channels were incorporated into the plasma membrane of Xenopus laevis oocytes after micro-injection of RNA from hen lower intestinal epithelium (colon and coprodeum). The animals were fed either a normal poultry food which contained NaCl (HS), or a similar food devoid of NaCl (LS). Oocytes were monitored for the expression of amiloride-sensitive sodium channels by measuring membrane potentials and currents. Oocytes injected with poly(A)+RNA prepared from HS animals or non-injected control oocytes showed no detectable sodium currents, whereas oocytes injected with LS-poly(A)+RNA had large amiloride-blockable sodium currents. These currents were almost completely saturated by sodium concentrations of 20 mM with a Km of about 2.6 mM sodium. Amiloride (10 microM) inhibits the expressed sodium channels entirely and examination of dose response relationships yielded a half-maximal inhibition concentration (Ki) of 120 nM amiloride. I-V difference curves in the presence or absence of sodium or amiloride (10 microM) indicate a potential dependence of the sodium transport which can be described by the Goldman equation. When Na+ is replaced by K+, no amiloride response was detected indicating a high selectivity for Na+ over K+. These results provide strong evidence that intestinal Na+ channels are regulated by dietary salt intake on the RNA level.

The kinetics of light-induced acidification and of the subsequent dark-induced alkalization in suspensions of sub-bacterial particles of Halobacterium halobium may be expressed as the sum of two exponentials, indicating two processes (Eisenbach, M., Bakker, E.P., Korenstein, R. and Caplan, S.R. (1976) FEBS Lett. 71, 228--232). We studied the effects of carbonyl cyanide p-trifluoromethyoxy phenyl-hydrazone, nigericin, gramicidin D, valinomycin, and monactin on the extents and the rate constants of the two processes. The various ionophores affected the two processes differently and in general the slower process was more sensitive to their presence. Valinomycin and monactin had relatively minor effects, apparently due to the high ionic strength of the suspension. When an artificial membrane potential was created in the dark, the light-induced acidification was preceded by a transient alkalization as is usually observed in intact cells. These results are discussed in the light of a suggested model accounting for the two processes (Caplan, S.R., Eisenbach, M., Cooper, S., Garty, H., Klemperer, G. and Bakker, E.P. (1977) in Bioenergetics of Membranes (Packer, L., Papageorgiou, G.C. and Trebst, A., eds.), pp. 101--114, Elsevier/North-Holland Biomedical Press, Amsterdam), taking into account the different selectivities of the ionophores applied.

Light-induced Na+ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na+ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na+ efflux, the inhibition being complete at low light intensity. The Na+ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na+ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na+ transport. Proton influx in the dark followed the artificial formation of a sodium gradient [Na+]in less than [Na+]out). These results may be explained by a Na+/H+ antiport mechanism. The fluxes of Na+ and H+ were of comparable magnitude, but the initial rate of Cl- efflux in the same experiment was one-third of the initial rate of Na+ efflux. Consequently Cl- is not regarded as a participant in the Na+ efflux mechanism.

Ouabain-blocked toad urinary bladders were maintained in Na+-free mucosal solutions, and a depolarizing solution of high K+ activity containing only 5 mM Na+ on the serosal side. Exposure to mucosal sodium (20 mM activity) evoked a transient amiloride-blockable inward current, which decayed to near zero within one hour. The apical sodium conductance increased in the initial phase of the current decay and decreased in the second phase. The conductance decrease required Ca2+ to be present on the serosal side and was more rapid when the mucosal Na+ activity was higher. At 20 mM mucosal Na+ and 3 mM serosal Ca2+ the initial (maximal) rate of inhibition amounted to 20% in 10 min. The conductance decrease could be accelerated by raising the serosal Ca2+ activity to 10 mM. The inhibition reversed on lowering the serosal Ca2+ to 3 microM and, in addition, the mucosal Na+ to zero. Exposure of the mucosal surface to the ionophore nystatin abolished the Ca2+ sensitivity of the transcellular conductance, showing that the Ca2+-sensitive conductance resides in the apical membrane. The data imply that in the K+-depolarized epithelia, cellular Ca2+, taken up from the serosal medium by means of a Na+-Ca2+ antiport, cause feedback inhibition by blockage of apical Na+ channels. However, the rate of inhibition is small, such that this regulatory mechanism will have little effect at 1 mM serosal Ca2+ and less than 20 mM cellular Na+.

The uptake of rubidium in intact Halobacterium halobium cells was followed, and found to be light-dependent. The exchange process is slow, the steady-state rate of 86Rb+/Rb+ exchange being given by k. = 6.3 - 10(-4) min-1. Starved cells exhibited a faster rate than unstarved cells. The influx of 86Rb+ was almost completely blocked in the presence of proton conductors (CCCP, FCCP, and SF 6847), and was sensitive to the presence of the permeant cation TPMP+. Valinomycin very slightly increased the rate of uptake, while 1 - 10(-6) M nigericin showed significant inhibition. On the other hand, release of 86Rb+ was not light-dependent, although still affected by uncouplers, TPMP+, and nigericin. These experimental observations may be explained in terms of a passive flux driven by an electrical potential difference, and influenced by positive isotope interaction within the membrane. In carefully matched influx-efflux studies, the extent of the positive isotope interaction was measured. Using the formal treatment of Kedem and Essig, the ratio (exchange resistance)/(resistance to net flow) for 86Rb+ was found to be 1.7.