Purinergic control of apical plasma membrane PI(4,5)P₂levels sets ENaC activity in principal cells

Abstract

Pochynyuk O, Bugaj V, Vandewalle A, Stockand JD. Purinergic control of apical plasma membrane PI(4,5)P 2 levels sets ENaC activity in principal cells. Am J Physiol Renal Physiol 294: F38-F46, 2008. First published October 3, 2007 doi:10.1152/ajprenal.00403.2007.-Activity of the epithelial sodium channel (ENaC) is limiting for Na ϩ reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5-biphosphate [PI(4,5)P 2] modulate the activity of this channel. Activation of purinergic receptors triggers multiple events, including activation of PKC and PLC, with the latter depleting plasma membrane PI(4,5)P 2. Here, we investigate regulation of ENaC in renal principal cells by purinergic receptors via PLC and PI(4,5)P 2. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)P 2 levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor U73122, but not U73343, its inactive analog, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting either purinergic receptors or PLC in resting cells dramatically increased ENaC activity above basal levels, indicating tonic activation of purinergic signaling in these polarized renal epithelial cells. Increased ENaC activity was associated with elevation of apical membrane PI(4,5)P 2 levels. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)P 2 levels. Dwell-time analysis reveals that depletion of PI(4,5)P 2 forces ENaC toward a closed state. In contrast, increasing PI(4,5)P 2 levels above basal values locks the channel in an open state interrupted by brief closings. Thus our results suggest that purinergic control of apical membrane PI(4,5)P 2 levels is a major regulator of ENaC activity in renal epithelial cells.phosphoinositides; PLC signaling; P2Y receptors; sodium reabsorption; hypertension THE EPITHELIAL NA ϩ CHANNEL (ENaC) is localized to the apical plasma membrane of many epithelia, including that in the aldosterone-sensitive distal renal nephron. The activity of this channel is limiting for Na ϩ (re)absorption across these epithelia (12,15,31,33,45). ENaC is a critical end-effector of the renin-angiotensin-aldosterone system. Thus ENaC is well positioned to regulate sodium homeostasis and, consequently, blood pressure. The physiological importance of ENaC to negative-feedback regulation of blood pressure is emphasized by inheritable forms of severe hypertension resulting from gain-of-function mutations in the channel (13,21,29,30,32).In contrast, loss-of-function mutations lead to improper salt wasting and low blood pressure (29,30).ENaC is a highly Na ϩ -selective, non-voltage-gated, noninactivating ion channel in the ENaC/Deg superfamily (3,12,15). This channel is heteromeric, comprising three distinct but similar subunits: ␣, ␤, and ␥ (7). Each subunit has NH 2 -and...

Purinergic signalling in the kidney in health and disease

“…Consistent with P2Y 2 receptors being the primary mediator of nucleotide-induced inhibition of ENaC, ATP's effects on sodium reabsorption were significantly reduced in collecting duct cells isolated from P2Y 2 −/ − receptor mice. However, residual ATP effects implicated the involvement of other P2 receptors [289]. Recently, P2Y 2 receptor activation has been shown to increase renal Na + excretion and decrease blood pressure [310].…”

“…The potentiating effect of apical P2X4/6 receptors (when luminal Na + is low) has not been investigated over a 30-min period, but a potentiating effect of basolaterally expressed P2X4-like receptors has been reported (reproduced from [414], with permission from Springer) sensitive distal nephron [309]. Resting ENaC activity was greater in P2Y 2 −/− receptor mice than controls suggesting that local ATP may be involved in the regulation of basal ENaC activity in the murine collecting duct [289,290]. Additional studies from the same laboratory demonstrated that ENaC downregulation in response to sodium restriction was lost in the P2Y 2 null mice, suggesting a role for the receptor in the renal response to altered sodium intake [291].…”

Thiazolidinedione-Induced Fluid Retention Is Independent of Collecting Duct αENaC Activity

“…Cell-attached patches were made under voltage-clamp conditions using pipettes having resistances of 10 to 15 megaohms on the apical plasma membranes of principal cells in isolated, split-open CDs and primary mouse IMCD cultures using standard procedures. 43,48,49 Bath and pipette solutions were as follows (in mM): 155 NaCl, 1 CaCl 2 , 2 MgCl 2 , 5 glucose, and 10 HEPES (pH 7.4) and 140 LiCl, 2 MgCl 2 , and 10 HEPES (pH 7.4), respectively. Single-channel current data from gigaohm seals were acquired (and subsequently analyzed) with an Axopatch 200B (Axon Instruments, Union City, CA) patchclamp amplifier interfaced via a Digidata 1322A (Axon Instruments) to a PC running the pClamp 9.2 suite of software (Axon Instruments).…”

Bestrophin 1 Promotes Epithelial-to-mesenchymal Transition of Renal Collecting Duct Cells

“…[1][2][3][4] In intact micro-perfused CDs, no evidence was found for a luminal Ca 2ϩ -dependent Cl -conductance (CaCC). 2,5 In contrast, CaCC was readily detectable in M1 mouse CD cells and primary cultured renal epithelial cells.…”