He-Ping Ma

Regulation of the epithelial sodium channel by phosphatidylinositides: experiments, implications, and speculations

Recent studies suggest that the activity of epithelial sodium channels (ENaC) is increased by phosphatidylinositides, especially phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Stimulation of phospholipase C by either adenosine triphosphate (ATP)-activation of purinergic P2Y receptors or epidermal growth factor (EGF)-activation of EGF receptors reduces membrane PI(4,5)P2, and consequently decreases ENaC activity. Since ATP and EGF may be trapped in cysts formed by the distal tubule, it is possible that ENaC inhibition induced by ATP and EGF facilitates cyst formation in polycystic kidney diseases (PKD). However, some results suggest that ENaC activity is increased in PKD. In contrast to P2Y and EGF receptors, stimulation of insulin-like growth factor-1 (IGF-1) receptor by aldosterone or insulin produces PI(3,4,5)P3, and consequently increases ENaC activity. The acute effect of aldosterone on ENaC activity through PI(3,4,5)P3 possibly accounts for the initial feedback for blood volume recovery after hypovolemic hypotension. PI(4,5)P2 and PI(3,4,5)P3, respectively, interacts with the N terminus of β-ENaC and the C terminus of γ-ENaC. However, whether ENaC selectively binds to PI(4,5)P2 and PI(3,4,5)P3 over other anionic phospholipids remains unclear.

Cyclosporine stimulates the renal epithelial sodium channel by elevating cholesterol

Cyclosporine A (CsA) is an efficient immunosuppressant used for reducing allograft rejection but with a severe side effect of causing hypertension. We hypothesize that the renal epithelial sodium channel (ENaC) may participate in CsA-induced hypertension. In the present study, we used the patch-clamp cell-attached configuration to examine whether and how CsA stimulates ENaC in A6 distal nephron cells. The data showed that CsA significantly increased ENaC open probability. Since CsA is an inhibitor of the ATP-binding cassette A1 (ABCA1) transporter, we employed 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), another ABCA1 inhibitor, and found that DIDS mimicked the effects of CsA on ENaC basal and cholesterol-induced activity but without any additive effect if combined with CsA. CsA and DIDS also had an identical effect on reduced ENaC activity caused by cholesterol extraction. ABCA1 protein was detected in A6 cells by Western blot analysis. Confocal microscopy data showed that both CsA and DIDS facilitated A6 cells to uptake cholesterol. Since enhanced ENaC activity is known to cause hypertension, these data together suggest that CsA may cause hypertension by stimulating ENaC through a pathway associated with inhibition of ABCA1 and consequent elevation of cholesterol in the cells.

WNK4 regulates the secretory pathway via which TRPV5 is targeted to the plasma membrane.

TRPV5 and TRPV6 are two closely related epithelial calcium channels that mediate apical calcium entry in the transcellular calcium transport pathway. TRPV5, but not TRPV6, is enhanced by protein kinase WNK4 when expressed in Xenopus laevis oocytes. We report that the majority of human TRPV5 exogenously expressed in the Xenopus oocyte plasma membrane was complexly N-glycosylated whereas that for human TRPV6 was core-glycosylated. Unglycosylated N358Q mutants of TRPV5 and TRPV6 were able to be expressed in the plasma membrane albeit with decreased abilities in mediating calcium uptake. Syntaxin 6, a SNARE protein in the trans-Golgi network, blocked the complex glycosylation of TRPV5 and TRPV6, rendered the channels in core-glycosylated form. Blocking complex glycosylation of TRPV5 either by syntaxin 6 or by N358Q mutation abolished the enhancing effect of WNK4 on TRPV5. Thus the difference in membrane expression of TRPV5 and TRPV6 explains the selective effect of WNK4 on TRPV5, which is likely on the secretory pathway involving complex glycosylation of channel proteins.

Role of the epithelial sodium channel in salt-sensitive hypertension.

The epithelial sodium channel (ENaC) is a heteromeric channel composed of three similar but distinct subunits, α, β and γ. This channel is an end-effector in the rennin-angiotensin-aldosterone system and resides in the apical plasma membrane of the renal cortical collecting ducts, where reabsorption of Na+ through ENaC is the final renal adjustment step for Na+ balance. Because of its regulation and function, the ENaC plays a critical role in modulating the homeostasis of Na+ and thus chronic blood pressure. The development of most forms of hypertension requires an increase in Na+ and water retention. The role of ENaC in developing high blood pressure is exemplified in the gain-of-function mutations in ENaC that cause Liddles syndrome, a severe but rare form of inheritable hypertension. The evidence obtained from studies using animal models and in human patients indicates that improper Na+ retention by the kidney elevates blood pressure and induces salt-sensitive hypertension.

An intermediate-conductance Ca 2+ -activated K + channel mediates B lymphoma cell cycle progression induced by serum

We have previously reported that Kv1.3 channel is expressed in Daudi cells. However, the present study demonstrates that Daudi cell cycle progression is not affected by margatoxin, a Kv1.3 channel blocker, but can be suppressed by tetraethylammonium (TEA) and 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), a selective blocker of intermediate-conductance Ca2+-activated K+ (IK) channels. Our patch-clamp data indicate that Daudi cells express an IK channel because it has a unit conductance of about 30xa0pS, is voltage-independent, and can be activated by submicromolar Ca2+ and blocked by TRAM-34. Fetal bovine serum (FBS) elevated intracellular Ca2+ concentration ([Ca2+]i) and activated this IK channel. Conversely, Rituximab, a human–mouse chimeric monoclonal antibody of CD20, significantly decreased [Ca2+]i and inhibited the channel. Furthermore, both FBS-induced IK channel expression and cell cycle progression were attenuated by the treatment with LY-294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. These data together suggest that a growth factor(s) in FBS triggers cell cycle progression by elevating both IK channel activity via CD20 and IK channel expression on the cell surface via PI3K. Thus, elevated IK channel activity and expression may account, in part, for Daudi cell malignant growth and proliferation.

Membrane tension modulates the effects of apical cholesterol on the renal epithelial sodium channel.

We used patch-clamp techniques and A6 distal nephron cells as a model to determine how cholesterol regulates the renal epithelial sodium channel (ENaC). We found that luminal methyl-β-cyclodextrin (mβCD, a cholesterol scavenger) did not acutely affect ENaC activity at a previously used concentration of 10 mm but significantly decreased ENaC activity both when the cell membrane was stretched and at a higher concentration of 50 mm. Luminal cholesterol had no effect on ENaC activity at a concentration of 50 μg/ml but significantly increased ENaC activity both when the cell membrane was stretched and at a higher concentration of 200 μg/ml. Confocal microscopy data indicate that membrane tension facilitates both mβCD extraction of cholesterol and A6 cell uptake of exogenous cholesterol. Together with previous findings that cholesterol in the apical membrane is tightly packed with sphingolipids and that stretch can affect lipid distribution, our data suggest that membrane tension modulates the effects of mβCD and cholesterol on ENaC activity, probably by facilitating both extraction and enrichment of apical cholesterol.

Incomplete Inactivation of Voltage-dependent K+ Channels in Human B Lymphoma Cells

The voltage-dependent K (KV) channel in Daudi human B lymphoma cells was characterized by using patch-clamp techniques. Whole-cell voltage-clamp experiments demonstrated that cell membrane depolarization induced a transient (time-dependent) outward current followed by a steady-state (time-independent) component. The time-dependent current resembled behavior of the type n channel, such as use dependence and a unique blockade by tetraethylammonium (TEA). Both time-dependent and time-independent currents were blocked by quinine with a similar IC50 (14.2 mM and 12.6 mM). Treatment with antisense oligonucleotide of human Kv1.3 gene significantly reduced both currents by 80%. Single-channel experiments showed that only one type of KV channel was recorded with a unitary conductance of approximately 19 pS. Consistent with whole-cell recordings, the channel activity in cell-attached patches remained in response to prolonged depolarization, and the remaining channel activity was blocked by quinine, but not TEA. Channel activity was scarcely seen in cell-attached patches after antisense treatment. Whole-cell current-clamp data showed that TEA, which blocks only the time-dependent current, caused a slight decrease in the membrane potential. In contrast, quinine and antisense, which block both time-dependent and -independent currents, strongly reduced the membrane potential. These data together suggest that the KV channel in Daudi cells does not completely inactivate and that the remaining channel activity due to this incomplete inactivation appears to be primarily responsible for maintaining the membrane potential.n

Hypertension and Sodium Channel Turnover

Hypertension is an increase of blood pressure to levels greater than normal that arises because of a mismatch between the volume of the vascular tree and the volume of blood. Blood volume depends on total body sodium content, which is a balance between sodium intake and output. Total body sodium is controlled by variable excretion of sodium by the kidneys. To regulate sodium balance, the primary variable that the kidney monitors is not total body sodium, but rather systemic blood pressure. Renal regulation of blood pressure is via the release of the peptide hormone, renin from specialized renal cells. Release of renin ultimately leads to the production of angiotensin II. Angiotensin II increases total peripheral resistance and blood pressure and also leads to an increase in aldosterone. Aldosterone is a steroid hormone that increases sodium reabsorption in the distal nephron by activating epithelial Na channels (ENaCs). Thus, Hypertension is a defect in one of these elements that control total body sodium balance.