Interaction with podocin facilitates nephrin signaling.

Mutations of NPHS1 or NPHS2, the genes encoding for the glomerular podocyte proteins nephrin and podocin, cause steroid-resistant proteinuria. In addition, mice lacking CD2-associated protein (CD2AP) develop a nephrotic syndrome that resembles NPHS mutations suggesting that all three proteins are essential for the integrity of glomerular podocytes. Although the precise glomerular function of either protein remains unknown, it has been suggested that nephrin forms zipper-like interactions to maintain the structure of podocyte foot processes. We demonstrate now that nephrin is a signaling molecule, which stimulates mitogen-activated protein kinases. Nephrin-induced signaling is greatly enhanced by podocin, which binds to the cytoplasmic tail of nephrin. Mutational analysis suggests that abnormal or inefficient signaling through the nephrin-podocin complex contributes to the development of podocyte dysfunction and proteinuria.

Characterisation of the rat SK4/IK1 K(+) channel.

BACKGROUND AND AIMS: The Ca(2+)-activated K(+) channel rSK4 is the rat homologue of the human SK4/IK1 (KCNN4) channel. In colonic mucosa rSK4 plays a key role during acetylcholin-induced secretion. This study was aimed to characterize the properties of the rat SK4 channel. METHODS: Electrophysiological measurements were performed on rSK4 expressing Xenopus laevis oocytes and rat colonic crypts. Intracellular Ca(2+) activity was assessed by Oregon Green fluorescence measurements. RESULTS: The 10 pS rSK4 expressed in oocytes was Ca(2+)-sensitive and inhibited by calmodulin antagonists. 1-ethyl-2-benzimidazolinone (1-EBIO), a known activator of SK4/IK1 channels, also activated rSK4. 1-EBIO affected the current neither at saturating Ca(2+) activities nor under Ca(2+)-free conditions, but increased the Ca(2+) sensitivity of rSK4. rSK4 was strongly activated by cytosolic ATP. However, PKA itself, PKA inhibitors and mutation of the PKA phosphorylation site (S332A) did not affect channel activity. The PKC activator 1,2-dioctanoyl-sn-glycerol and the PKC inhibitor bisindolylmaleimide also failed to influence rSK4. CONCLUSION: The Ca(2+)-sensitive rSK4 is activated by 1-EBIO probably via facilitation of the Ca(2+)-calmodulin-rSK4 interaction. The strong ATP-activation of rSK4 is likely to be caused by phosphorylation via a yet unknown kinase and might involve additional subunits.

Carbachol activates a K+ channel of very small conductance in the basolateral membrane of rat pancreatic acinar cells.

Secretion of Cl- requires the presence of a K+ conductance to hyperpolarize the cell, and to provide the driving force for Cl- exit via luminal Cl- channels. In the exocrine pancreas Cl- secretion is mediated by an increase in cytosolic Ca2+ ([Ca2+]i). Two types of Ca2+-activated K+ channels could be shown in pancreatic acinar cells of different species. However, there are no data on Ca2+-activated K+ channels in rat pancreatic acini. Here we examine the basolateral K+ conductance of freshly isolated rat pancreatic acinar cells in cell-attached and cell-excised patch-clamp experiments. Addition of carbachol (CCH, 1 micromol/l) to the bath led to the activation of very small conductance K+ channels in cell-attached patches (n=27), producing a noisy macroscopic outward current. The respective outward conductance increased significantly by a factor of 2.1+/-0.1 (n=27). Noise analysis revealed a Lorentzian noise component with a corner frequency (f(c)) of 30.3+/-3.5 Hz (n=19), consistent with channel activity in these patches. The estimated single-channel conductance was 1.5+/-0.4 pS (n=19). In cell-excised patches (inside out) from cells previously stimulated with CCH, channel activity was only observed in the presence of K+ in the bath solution. Under these conditions f(c) was 47.6+/-11.9 Hz (estimated single-channel conductance 1.1+/-0.2 pS, n=20). The current/voltage relationship of the noise showed weak inward rectification and the reversal potential shifted towards E(K+) when Na+ in the bath was replaced by K+. Channel activity in cell-excised patches was slightly reduced by 10 mmol/l Ba2+ (23.6+/-2.1% of the total outward current) and was completely absent when K+ in the bath was replaced by Na+. Reduction of the [Ca2+]i from 1 mmol/l to 1 micromol/l in cell-excised experiments decreased the current by 52.3+/-12.3% (n=5). Expression of K(v)LQT1, one of the possible candidates for a small-conductance K+ channel in rat pancreatic acinar cells, was shown by reverse transcriptase polymerase chain reaction (RT-PCR). In fact, a K(V)LQT-blocker (chromanol 293B) reduced channel activity in seven excised patches. These data suggest that CCH activates very small conductance K+ channels in rat pancreatic acinar cells, most likely via an increase in [Ca2+]i.

N-Acetyl-L-cysteine and its derivatives activate a Cl- conductance in epithelial cells.

N-Acetyl-L-cysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders including cystic fibrosis (CF). The beneficial effects of NAC are empirical and the exact mechanism of action in the airways remains obscure. In the present study we examined the effects on whole-cell (wc) conductance (Gm) and voltage (Vm) of NAC and the congeners S-carboxymethyl-L-cysteine (CMC) and S-carbamyl-L-cysteine (CAC) and L-cysteine in normal and CF airway epithelial cells. L-Cysteine (1 mmol/l) had no detectable effect. The increase in Gm (delta Gm) by the other compounds was concentration dependent and was (all substances at 1 mmol/l) 3.8 +/- 1.4 nS (NAC; n = 11), 4.2 +/- 1.0 nS (CMC; n = 16) and 3.8 +/- 1.6 nS (CAC; n = 18), respectively. The changes in Gm were paralleled by an increased depolarization (delta Vm) when extracellular Cl- concentration was reduced to 34 mmol/l: under control conditions = -4.1 +/- 2.1 versus 10.2 +/- 2.1 mV in the presence of NAC, CMC, CAC (n = 36). In the presence of NAC, CMC and CAC, the reduction in Cl- concentration was paralleled by a reduction of Gm by 2.1 +/- 0.4 nS (n = 35), indicating that all substances acted by increasing the Cl- conductance. Analysis of intracellular pH did not reveal any changes by any of the compounds (1 mmol/l). A Cl- conductance was also activated in HT29 colonic carcinoma and CF tracheal epithelial (CFDE) cells but not in CFPAC-1 cells, which do not express detectable levels of delta F508-CFTR, suggesting that the presence of CFTR may be a prerequisite for the induction of Cl- currents. Next we examined the ion currents in Xenopus oocytes microinjected with CFTR-cRNA. Water-injected oocytes did not respond to activation by forskolin and 3-isobutyl-1-methylxanthine (IBMX) (delta Gm = 0.08 +/- 0.04 microS; n = 10) and no current was activated when these oocytes were exposed to NAC or CMC. In contrast, in CFTR-cRNA-injected oocytes Gm was enhanced when intracellular adenosine 3',5'-cyclic monophosphate (cAMP) was increased by forskolin and IBMX (Gm = 4.5 +/- 1.3 microS; n = 8). Gm was significantly increased by 0.74 +/- 0.2 microS (n = 11) and 0.46 +/- 0.1 microS (n = 10) when oocytes were exposed to NAC and CMC, respectively (both 1 mmol/l). In conclusion, NAC and its congeners activate Cl- conductances in normal and CF airway epithelial cells and hence induce electrolyte secretion which may be beneficial in CF patients. CFTR appears to be required for this response in an as yet unknown fashion.

Effect of NH4+/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop.

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henle's loop of rat kidney (TAL) are dominated by K+. In excised membrane patches the luminal K+ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (Vm) in slow-whole-cell (SWC) recordings and the open probability (Po) of K+ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of Vm can be recorded. The pipette solution contained K+ 125 mmol/l and Cl- 32 mmol/l. Intracellular pH was determined by 2',7'bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH4+/NH3 to the bath. In the presence of NH4+/NH3 intracellular pH acidified by 0.53 +/- 0.11 units (n = 7). Inhibition of the Na+2Cl-K+ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62 +/- 0.14 units (n = 7). In SWC experiments Vm of TAL cells was -72 +/- 1 mV (n = 70). NH4+/NH3 depolarised Vm by 22 +/- 2 mV (n = 25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH4+ from 24 +/- 3 mV to 7 +/- 3 mV. Under control conditions the single-channel conductance of TAL K+ channels in CAN experiments was 66 +/- 5 pS and the reversal voltage for K+ currents was 70 +/- 2 mV (n = 35). The Po of K+ channels in CAN patches was reduced by NH4+/NH3 from 0.45 +/- 0.15 to 0.09 +/- 0.07 (n = 7). NH4+/NH3 exposure depolarised the zero current voltage of the permeabilised patches by -9.7 +/- 3.6 mV (n = 5). The results show that TAL K+ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH4+/NH3 exposure at concentrations which are physiologically relevant because Na+2Cl-K+(NH4+) cotransporter-mediated import of NH4+ exceeds the rate of NH3 diffusion into the TAL. K+ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH4+ uptake occurs by the Na+2Cl-K+ cotransporter. The findings that, in the presence of NH4+/NH3 and furosemide, Vm is not completely repolarised and that K+ channels are not activated suggest that the respective K+ channels may in addition to their pH regulation be inhibited directly by NH4+/NH3.

pH regulation in HT29 colon carcinoma cells.

The pH regulation in HT29 colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pHi) was 7.21 +/- 0.07 (n = 22) in HCO3(-)-containing and 7.21 +/- 0.09 (n = 12) in HCO3(-)-free solution. HOE-694 (10 mumol/l), a potent inhibitor of the Na+/H+ exchanger, did not affect control pHi. As a means to acidify cells we used the NH4+/NH3 (20 mmol/l) prepulse technique. The mean peak acidification was 0.37 +/- 0.07 pH units (n = 6). In HCO3(-)-free solutions recovery from acid load was completely blocked by HOE-694 (1 mumol/l), whereas in HCO3(-)-containing solutions a combination of HOE-694 and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na(+)-dependent in HCO3(-)-containing and HCO3(-)-free solutions. Removal of external Cl- caused a rapid, DIDS-blockable alkalinization of 0.33 +/- 0.03 pH units (n = 15) and of 0.20 +/- 0.006 pH units (n = 5), when external Na+ was removed together with Cl-. This alkalinization was faster in HCO3(-)-containing than in HCO3(-)-free solutions. The present observations demonstrate three distinct mechanisms of pHi regulation in HT29 cells: (a) a Na+/H+ exchanger, (b) a HCO3-/Cl- exchanger and (c) a Na(+)-dependent HCO3- transporter, probably the Na(+)-HCO3-/Cl- antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)