Permsak Komwatana

All Three WW Domains of Murine Nedd4 Are Involved in the Regulation of Epithelial Sodium Channels by Intracellular Na

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of α, β, and γ subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle’s syndrome where mutations delete or alter the PY motif of either the β or γ subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind α-, β-, and γ-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+channel and of WW domain 1 to an unknown associated protein.

Control of the amiloride‐sensitive Na+ current in mouse salivary ducts by intracellular anions is mediated by a G protein.

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.

Activators of epithelial Na+ channels inhibit cytosolic feedback control. Evidence for the existence of a G protein-coupled receptor for cytosolic Na+.

Abstract. We have previously shown that epithelial Na+ channels in mouse mandibular gland duct cells are controlled by cytosolic Na+ and Cl−, acting, respectively, via Go and Gi 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 Go in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na+ receptor.

Characterization of the Cl- conductance in the granular duct cells of mouse mandibular glands.

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 μmol/l), 4,4′-diisothiocyanato-stilbene-2,2′-disulphonate (DIDS, 100 μmol/l), indanyloxyacetic acid (IAA-94, 100 μmol/l), verapamil (100 μmol/l), glibenclamide (100 μmol/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.

Control of the Amiloride-Sensitive Na+ Current in Salivary Duct Cells by Extracellular Sodium

Abstract.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 Kmof 47.3 mmol/1 and a maximum conductance (Gmax) 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.

Roles of the C Termini of α-, β-, and γ-Subunits of Epithelial Na+ Channels (ENaC) in Regulating ENaC and Mediating Its Inhibition by Cytosolic Na+

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 Na+. 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 α-, β-, and γ-subunits of ENaC in mediating this inhibition. We found that peptides corresponding to the C termini of the β- and γ-subunits, but not the α-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 β-ENaC, the interacting motif included βSer631, βAsp632, and βSer633. In the C terminus of mouse γ-ENaC, it included γSer640. Once these motifs were deleted, we were able to use the C termini of β- and γ-ENaC to prevent Nedd4-mediated down-regulation of Na+ channel activity. The C terminus of the α-subunit, on the contrary, did not prevent Nedd4-mediated inhibition of the Na+ channels. We conclude that mouse Nedd4 interacts with the β- and γ-subunits of ENaC.

Patch-clamp studies on epithelial sodium channels in salivary duct cells

Mouse mandibular salivary duct cells contain an amiloride-sensitive Na+ current and express all three subunits of the epithelial Na+ channel, ENaC. This amiloride-sensitive Na+ current is subject to feedback regulation by intracellular Na+ and we have previously demonstrated that this regulation is mediated by an ubiquitin-protein ligase, which we identified as Nedd4. The evidence supporting this identification is as follows: (1) antibodies raised against murine Nedd4 block Na+ feedback inhibition; (2) a mutant of murine Nedd4 containing the WW domains but no HECT domain (ubiquitin-protein ligase) blocks Na+ feedback inhibition; and (3) Nedd4 is expressed in mouse mandibular salivary duct cells.In the present studies, we have used whole-cell patch-clamp methods to further investigate the mechanisms by which ubiquitin-protein ligases regulate the amiloride-sensitive Na+ conductance in mouse salivary duct cells. In particular, we have examined the possibility that the ubiquitin-protein which ubiquitin-protein ligases inhibit Na+ channels.We have found that KIAA0439 is expressed in mouse mandibular ducts and interacts with the PY motifs of the α-, β-, and γ-subunits of ENaC in vitro. Furthermore, in whole-cell patch-clamp studies, a glutathione-S-transferase (GST)-fusion protein containing the WW motifs of human KIAA0439 was able to inhibit feedback regulation of the amiloride-sensitive Na+ current by intracellular Na+. We also examined whether GST-fusion proteins containing the C-termini of the α-, β-, and γ-subunits of ENaC are able to interrupt Na+ feedback regulation of the amiloride-sensitive Na+ current. We found that the C-termini of the β- and γ-subunits were able to do so, whereas the C-terminus of the α-subunit was not.We conclude that KIAA0439 is, together with Nedd4, a potential mediator of the control of epithelial Na+ channels in salivary duct cells by intracellular Na+. We further conclude that ubiquitin-protein ligases interact with the Na+ channels through the C-termini of the β- and γ-subunits of the Na+ channels.

Osmotic Sensitivity of the Hyperpolarization-Activated Cl– Current in Mouse Mandibular Duct Cells

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 C1C-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 C1C-2 channels, perhaps due to a difference in the mechanism by which the osmolality signal is transduced to a change in channel activity.

Intracellular Ca2+ inactivates an outwardly rectifying K+ current in human adenomatous parathyroid cells

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.