Epithelial Na + channels mediate the transport of Na across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, with loss of this inhibition leading to hypertension. Here, we report that these channels are maintained in the active state by the G protein-coupled receptor kinase, Grk2, which has been previously implicated in the development of essential hypertension. We also show that Grk2 phosphorylates the C terminus of the channel β subunit and renders the channels insensitive to inhibition by Nedd4-2. This mechanism has not been previously reported to regulate epithelial Na + channels and provides a potential explanation for the observed association of Grk2 overactivity with hypertension. Here, we report a G protein-coupled receptor kinase regulating a membrane protein other than a receptor and provide a paradigm for understanding how the interaction between membrane proteins and ubiquitin protein ligases is controlled.

The amiloride-sensitive epithelial Na+ channels (ENaC) in the intralobular duct cells of mouse mandibular glands are inhibited by the ubiquitin-protein ligase, Nedd4, which is activated by increased intracellular Nat. In this study we have used whole-cell patch clamp methods in mouse mandibular duct cells: to investigate the role of the C termini of the alpha-, beta-, and gamma -subunits of ENaC in mediating this inhibition. We found that peptides corresponding to the C termini of the beta- and gamma -subunits, but not the alpha -subunit, inhibited the activity of the Na+ channels. This mechanism did not involve Nedd4 and probably resulted from the exogenous C termini interfering competitively with the protein-protein interactions that keep the channels active. In the case of the C terminus of mouse beta -ENaC, the interacting motif in eluded beta Ser(631), beta Asp(632) and beta Ser(633). In the C terminus of mouse gamma -ENaC, it included gamma Ser(640). Once these motifs were deleted, we were able to use the C termini of beta- and gamma -ENaC to prevent Nedd4-mediated down-regulation of Na+ channel activity. The C terminus of the alpha -subunit, on the contrary, did not prevent Nedd4-mediated inhibition of the Na+ channels. We conclude that mouse Nedd4 interacts with the beta- and gamma -subunits of ENaC.

It recently has been shown that epithelial Na(+) channels are controlled by a receptor for intracellular Na(+), a G protein (G(o)), and a ubiquitin-protein ligase (Nedd4). Furthermore, mutations in the epithelial Na(+) channel that underlie the autosomal dominant form of hypertension known as Liddle's syndrome inhibit feedback control of Na(+) channels by intracellular Na(+). Because all epithelia, including those such as secretory epithelia, which do not express Na(+) channels, need to maintain a stable cytosolic Na(+) concentration ([Na(+)](i)) despite fluctuating rates of transepithelial Na(+) transport, these discoveries raise the question of whether other Na(+) transporting systems in epithelia also may be regulated by this feedback pathway. Here we show in mouse mandibular secretory (endpiece) cells that the Na(+)-H(+) exchanger, NHE1, which provides a major pathway for Na(+) transport in salivary secretory cells, is inhibited by raised [Na(+)](i) acting via a Na(+) receptor and G(o). This inhibition involves ubiquitination, but does not involve the ubiquitin protein ligase, Nedd4. We conclude that control of membrane transport systems by intracellular Na(+) receptors may provide a general mechanism for regulating intracellular Na(+) concentration.

We have previously shown that epithelial Na+ channels in mouse mandibular gland duct cells are controlled by cytosolic Na+ and Cl−, acting, respectively, via G o and G i proteins. Since we found no evidence for control of epithelial Na+ channels by extracellular Na+ ([Na+] o ), our findings conflicted with the long-held belief that Na+ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues, like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel site which otherwise inhibits channel activity in response to increasing [Na+] o . Instead, we now show that PCMPS acts by rendering epithelial Na+ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na+ and Cl− on Na+ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane, block feedback inhibition of Na+ channels by cytosolic Na+, while leaving inhibition by cytosolic Cl− unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated α-subunit of G o in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na+ receptor.

Epithelial Na+ channels are expressed widely in absorptive epithelia such as the renal collecting duct and the colon and play a critical role in fluid and electrolyte homeostasis. Recent studies have shown that these channels interact via PY motifs in the C terminals of their alpha, beta, and gamma subunits with the WW domains of the ubiquitin-protein ligase Nedd4. Mutation or deletion of these PY motifs (as occurs, for example, in the heritable form of hypertension known as Liddle's syndrome) leads to increased Na+ channel activity. Thus, binding of Nedd4 by the PY motifs would appear to be part of a physiological control system for down-regulation of Na+ channel activity. The nature of this control system is, however, unknown. In the present paper, we show that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. We further show that Nedd4 operates downstream of Go in this feedback pathway. We find, however, that Nedd4 is not involved in the feedback control of Na+ channels by intracellular anions. Finally, we show that Nedd4 has no influence on Na+ channel activity when the Na+ and anion feedback systems are inactive. We conclude that Nedd4 normally mediates feedback control of epithelial Na+ channels by intracellular Na+, and we suggest that the increased Na+ channel activity observed in Liddle's syndrome is attributable to the loss of this regulatory feedback system.

The duct cells of the mandibular glands of mice (and many other mammalian salivary glands) absorb NaCl from an isotonic, Na+-rich primary saliva, formed by the gland's secretory endpieces, utilising an amiloride-sensitive Na+ channel in the apical (luminal) domain of the plasma membranes. The present study focuses on the mechanisms whereby the apical membrane Na+ conductance is controlled so that the rate of Na+ influx from lumen to cytosol via the Na+ channels is matched to the rate of Na+ extrusion from cytosol to interstitium via the basolateral Na+-K+-ATPase (so called homocellular regulation or epithelial cross-talk). Our results show that the apical membrane Na+ conductance is not controlled by a sensor of extracellular (luminal) Na+, as has been previously believed, but by sensors of cytosolic Na+ and Cl- which down-regulate the Na+ channels when the cytosolic concentration of either ion increases. These effects of cytosolic Na+ and Cl- are mediated, respectively, by G proteins of the Gi and Go subclasses.

1. We used microspectrofluorimetry with the pH-sensitive fluoroprobe 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) to study the regulation of cytosolic pH (pHi) in the isolated, perfused main excretory duct of the mouse mandibular gland. 2. In nominally HCO3(-)-free solutions, removal of Na+ from the lumen alone caused pHi to decline whereas removing it from the bath alone did not. 3. Readmission of Na+ to the lumen of ducts studied under zero-Na+ conditions caused pHi to recover fully. This recovery was blocked by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) with a half-maximum concentration of 0.5 mumol l-1, indicating the presence of an apical Na(+)-H+ exchanger. 4. Readmission of Na+ to the bath of ducts studied under zero-Na+ conditions also caused pHi to recover. This recovery was blocked by 100 mumol l-1 EIPA, indicating the presence of a basolateral Na(+)-H+ exchanger. 5. Measurements of H+ fluxes indicated that the apical Na(+)-H+ exchanger was approximately four times more active than the basolateral Na(+)-H+ exchanger. 6. In three sets of experiments (in the absence of Na+, in the presence of Na+, and in the presence of Na+ plus 100 mumol l-1 EIPA), the effects of changing luminal K+ concentration on pHi were examined. We found no evidence for the presence of K(+)-H+ exchange or Na(+)-coupled K(+)-H+ exchange in the apical membranes of duct cells. 7. pHi recovery under nominally HCO3(-)-free conditions following acidification with an NH4Cl pulse was abolished by removal of Na+ from the bath and luminal solutions, indicating that no Na(+)-independent systems such as H(+)-ATPases were present. 8. A repeat of the above experiments in the presence of 25 mmol l-1 HCO3- plus 5% CO2 did not reveal any additional H+ transport systems. The removal of luminal Cl-, however, caused a small rise in pHi. This latter effect was blocked by 500 mumol l-1 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (H2-DIDS), suggesting that a Cl(-)-HCO3- exchanger in the apical membrane might contribute in a minor way to pHi regulation. 9. We conclude that the predominant H+ transport systems in the mouse mandibular main excretory duct are Na(+)-H+ exchangers in the apical and the basolateral membranes. The model we postulate to account for electrolyte transport across the main duct in the mouse mandibular gland is quite different from that previously developed for the rat duct but is similar to that developed for the rabbit duct. The difference is in concordance with the known ability of the mandibular gland of the rat, but not the rabbit or the mouse, to secrete a HCO3(-)-rich final saliva.

Although acetylcholine (ACh) is able to activate voltage- and Ca2+-sensitive K+ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole cell K+ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K+ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was sensitive to quinine and it conducted Rb+ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels. When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K+ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K+ current is not carried substantially by BK channels and show that it is carried by a small-conductance K+ channel with quite different properties.

In tight Na+-absorbing epithelial cells, the fate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-beta-S, pertussis toxin, and antibodies against the alpha-subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na+ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na+ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na+, we decided to use whole-cell patch clamp techniques to investigate whether the Na+ conductance in salivary duct cells is also regulated by extracellular Na+. Using Na+-free pipette solutions, we observed that the whole-cell Na+ conductance increased when the extracellular Na+ was increased, whereas the whole-cell Na+ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na+ conductance on extracellular Na+ could be described by the Michaelis-Menten equation with a K m of 47.3 mmol/1 and a maximum conductance (G max) of 2.18 nS. To investigate whether this saturation of the Na+ conductance with increasing extracellular Na+ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of the noise generated during the onset of blockade of the Na+ current with 200 μmol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na+ solutions. We found that Na+ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing extracellular Na+. On the other hand, the single-channel current saturated with increasing extracellular Na+ and, consequently, whole-cell Na+ permeability declined. In other words, the decline in Na+ permeability in salivary duct cells with increasing extracellular Na+ concentration is due simply to saturation of the single-channel Na+ conductance rather than to inactivation of channel activity.

We have previously shown that unstimulated mouse mandibular granular duct cells contain a hyperpolarization-activated Cl- conductance having characteristics similar to those of ClC-2 channels. We now show that exposure of the granular duct cells to hypotonic solutions inhibits the hyperpolarization-activated Cl- current whereas exposure to hypertonic solutions causes it to be transiently activated and then inhibited. Like ClC-2 channels in Xenopus oocytes, the hyperpolarization-activated Cl- current in mouse granular duct cells is sensitive to osmolality, although its response to osmolality is the reverse of that seen with ClC-2 channels, perhaps due to a difference in the mechanism by which the osmolality signal is transduced to a change in channel activity.

1. We have previously reported that the Na+ conductance in mouse intralobular salivary duct cells is controlled by cytosolic anions, being inhibited by high cytosolic concentrations of Cl‐ and NO3‐ but not of glutamate. In the present paper, we use whole‐cell patch‐clamp methods to investigate whether this anion effect is mediated by a G protein. 2. Inclusion of 100 mumol l‐1 GTP‐gamma‐S, a non‐hydrolysable GTP analogue, in the glutamate‐containing pipette solution, i.e. when the Na+ conductance is active, reduced the size of the Na+ conductance whereas inclusion of 100 mumol l‐1 GDP‐beta‐S, a non‐hydrolysable GDP analogue, had no effect. 3. Inclusion of 100 mumol l‐1 GDP‐beta‐S in the NO3(‐)‐containing pipette solution, i.e. when the Na+ conductance is inhibited, reactivated the conductance. Inclusion of 500 ng ml‐1 activated pertussis toxin in the NO3(‐)‐containing pipette solution had a similar effect on the Na+ conductance. 4. We conclude that the inhibitory effect of intracellular anions such as NO3‐ and Cl‐ on the amiloride‐sensitive Na+ conductance in mouse mandibular intralobular duct cells is mediated by a G protein sensitive to pertussis toxin.

We have previously shown that mouse mandibular granular ducts contain a hyperpolarization-activated Cl- conductance. We now show that the instantaneous current/voltage (I/V) relation of this Cl- conductance is inwardly rectifying with a slope conductance of 15.4 +/- 1.8 nS (n = 4) at negative potentials and of 6.7 +/- 0.9 nS (n = 4) at positive potentials. Thus, the inward rectification seen in the steady-state I/V relation is due, not only to voltage activation of the Cl- conductance, but also to the intrinsic conductance properties of the channel. We show further that the ductal Cl- conductance is not activated by including ATP (10 mmol/l) in the pipette solution. Finally, we show that the conductance is not blocked by the addition of any of the following compounds to the extracellular solution: anthracene-9-carboxylate (A9C, 1 mmol/l), diphenylamine-2-carboxylate (DPC, 1 mmol/l), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 mumol/l), 4,4'-diisothiocyanato-stilbene-2,2'-disulphonate (DIDS, 100 mumol/l), indanyloxyacetic acid (IAA-94, 100 mumol/l), verapamil (100 mumol/l), glibenclamide (100 mumol/l) and Ba2+ (5 mmol/l). The properties of the ductal Cl- conductance most nearly resemble those of the ClC-2 channel. Both channel types have instantaneous I/V relations that are slightly inwardly rectifying, are activated by hyperpolarization with a time-course in the order of hundreds of milliseconds, have a selectivity sequence of Br- > Cl- > I-, and are insensitive to DIDS. The only identified difference between the two is that the ClC-2 channel is 50% blocked both by DPC and A9C (1 mmol/l), whereas the ductal Cl- conductance is insensitive to these compounds.

We have used single-channel patch-clamp techniques to study the ion channels in the basolateral membranes of intralobular duct cells from the mouse mandibular gland. In 39% of cell-attached patches, we observed a K+ channel that had an inwardly rectifying current/voltage (I/V) relation with a maximum slope conductance of 123±9 pS (n=12) and a zero current potential of +49.4±3.4 mV (n=5) relative to the resting cell potential. The selectivity sequence of this channel, as estimated by zero current potential measurements, was: K+ (1) > Rb+ (0.38) > NH 4 + (<0.34), Cs+ (<0.16) > Na+ (<0.028). The activity of the channel was not affected by changes in membrane potential, nor was it affected by changes in the free Ca2+ concentration on the cytosolic side of inside-out excised patches in the range 1 nmol/l to 1 μmol/l. In 38% of cell-attached patches we observed a second K+ channel type with a maximum slope conductance of 62±3 pS (n=12) and an inwardly rectifyingI/V relation. The selectivity sequence of this channel was K+ (1) > Rb+ (<0.5) > NH 4 + (<0.2) > Na+ (<0.09). The activity of this channel type was not affected by changes in membrane potential. In 18% of excised patches, we also observed a non-selective cation channel that was not demonstrable in cell-attached patches. It had a slope conductance of 22±2 pS (n=6) and was blocked by the non-selective cation channel blocker, flufenamate (10 μmol/l). A fourth channel type, observed only in 5% of patches was a Cl− channel with a slope conductance of 40 pS and a linearI/V relation. The K+ channels observed in this study seem likely to underlie the K+ conductance described in the basolateral membrane of extralobular ducts by in vitro perfusion studies. Our finding that they are inwardly rectifying suggests that they may not be the sole route of K+ transport across the basolateral membrane.

We have used whole-cell patch-clamp techniques to study the conductances in the plasma membranes of human parathyroid cells. With a KCl-rich pipette solution containing Ca2+ buffered to a concentration of 0.1 μmol/l, the zero current potential was −71.1±0.5 mV (n=19) and the whole-cell current/ voltage (I/V) relation had an inwardly rectifying and an outwardly rectifying component. The inwardly rectifying current activated instantaneously on hyperpolarization of the plasma membrane to potentials more negative than −80 mV, and a semi-logarithmic plot of the reversal potential of the inward current (estimated by extrapolation from the range in which it was linear) as a function of extracellular K+ concentration ([K+]o) revealed a linear relation with a slope of 64 mV per decade change in [K+]o, which is not significantly different from the Nernstian slope, demonstrating that the current was carried by K+ ions. The conductance exhibited a square root dependence on [K+]o as has been observed for inward rectifiers in other tissues. The current was blocked by the presence of Ba2+ (1 mmol/l) or Cs+ (1.5 mmol/l) in the bath. The outwardly rectifying current was activated by depolarization of the membrane potential to potentials more positive than −20 mV. It was inhibited by replacement of pipette K+ with Cs+, indicating that it also was a K+ current: it was partially (42%) blocked when tetraethylammonium (TEA+, 10 mmol/l) was added to the bath. The outwardly rectifying, but not the inwardly rectifying K+ current, was regulated by intracellular free Ca2+ concentration ([Ca2+]i) such that increasing [Ca2+]i above 10 nmol/l inhibited the outwardly rectifying current, the half-maximum effect being seen at 1 μmol/l. Since it is known that increases in [Ca2+]o produce increases in [Ca2+]i, and that they depolarize parathyroid cells by reducing the membrane K+ conductance, we suggest that it is the reduction of the outwardly rectifying K+ conductance by increases in [Ca2+]i which is responsible for the reduction in K+ conductance seen when [Ca2+]o is increased.

Our previously published whole-cell patch-clamp studies on the cells of the intralobular (granular) ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na+ conductance in the plasma membrane. In this study we demonstrate the presence also of a Cl− conductance and we show that the sizes of both conductances vary with the Cl− concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl− concentration rises from 5 to 150 mmol/liter, the size of the inward Na+ current declines, the decline being half-maximal when the Cl− concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl− concentration increases, the inward Cl− current remains at a constant low level until the Cl− concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl− in the pipette solution was replaced by other anions indicate that the Na+ current is suppressed by intracellular Br-, Cl− and NO 3 - but not by intracellular I-, glutamate or gluconate. Our studies also show that the Cl− conductance allows passage of Cl− and Br- equally well, I-less well, and NO 3 - , glutamate and gluconate poorly, if at all. The findings with NO 3 - are of particular interest because they show that suppression of the Na+ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl− conductance. In mouse granular duct cells there is, thus, a reciprocal regulation of Na+ and Cl− conductances by the cytosolic Cl− concentration. Since the cytosolic Cl− concentration is closely correlated with cell volume in many epithelia, this reciprocal regulation of Na+ and Cl− conductances may provide a mechanism by which ductal Na+ and Cl transport rates are adjusted so as to maintain a stable cell volume.

Whole-cell patch clamp studies on dispersed cells from the intralobular ducts of mouse mandibular glands reveal the presence of an amiloride-sensitive Na(+)-selective conductance that is not found in the cells of the secretory endpieces. Since studies on ripped-off outside-out patches from the basolateral membranes of isolated intralobular ducts show that the membrane conductance is not altered by amiloride, it can be inferred that the Na+ conductance seen in whole-cell experiments is located in the luminal membrane. These studies thus provide evidence for the presence of a luminal Na+ conductance in salivary ducts and indicate that intralobular and extralobular ducts may handle Na+ in a similar manner.

The ion channel populations of cells prepared from human adenomatous parathyroid glands have been investigated using standard patch-clamp techniques with pipettes containing isotonic K+-rich solutions. The principal channel type observed was an inwardly rectifying K+ channel of 35 pS conductance. In addition, we have frequently observed a lower conductance (12 pS) K+ channel that appeared to conduct current in both directions. In addition to these K+ channels, we have observed non-selective cation channels and possibly Cl− channels. Although we have observed occasional current transitions that might have arisen from a large conductance K+ channel similar to that observed in rat and bovine parathyroid cells, we have not been able positively to identify such a channel in human parathyroid cells, either in cell-attached or in excised inside-out patches.

We have used microspectrofluorimetry with the pH-sensitive dye, BCECF, to examine the control of intracellular pH in the secretory endpieces of the sheep parotid gland. Unstimulated endpieces in HCO3--free media have a cytosolic pH of 7.5 +/- 0.03 (n = 69) which is maintained by a Na+-dependent proton extrusion process that can be partially supported by Li+ but not by Cs+, and is not affected by changes in extracellular Cl-. HCO3- or K+. It is not blocked by SITS or DIDS, which inhibit Na+-(n)HCO3- co-transport and Cl--HCO3-, exchange, nor is it sensitive to the amiloride analogs, MIA and EIPA, which inhibit Na+-H+ exchangers, although very high concentrations of amiloride itself (1 mmol/l) have a (probably non-specific) inhibitory effect. It seems likely that sheep parotid secretory endpieces do contain a Na+-H+ exchanger that drives secretion of a HCO3--rich juice, and that its insensitivity to amiloride and its analogs explains why these drugs do not block fluid secretion by the intact sheep parotid gland.