Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion.

Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk(-/-) mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1(-/-) mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1(+/+) and Romk1(-/-) mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1(+/+), but such regulation by high K intake was diminished with significant hyperkalemia in Romk1(-/-) mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K(+) secretion in the collecting tubule.

Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA-regulated apical chloride channels in cortical collecting duct.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK.

Substrate specificity of Rhbg: ammonium and methyl ammonium transport.

Rhbg is a nonerythroid membrane glycoprotein belonging to the Rh antigen family. In the kidney, Rhbg is expressed at the basolateral membrane of intercalated cells of the distal nephron and is involved in NH4+ transport. We investigated the substrate specificity of Rhbg by comparing transport of NH3/NH4+ with that of methyl amine (hydrochloride) (MA/MA+), often used to replace NH3/NH4+, in oocytes expressing Rhbg. Methyl amine (HCl) in solution exists as neutral methyl amine (MA) in equilibrium with the protonated methyl ammonium (MA+). To assess transport, we used ion-selective microelectrodes and voltage-clamp experiments to measure NH3/NH4+- and MA/MA+-induced intracellular pH (pH(i)) changes and whole cell currents. Our data showed that in Rhbg oocytes, NH3/NH4+ caused an inward current and decrease in pH(i) consistent with electrogenic NH4+ transport. These changes were significantly larger than in H2O-injected oocytes. The NH3/NH4+-induced current was not inhibited in the presence of barium or in the absence of Na+. In Rhbg oocytes, MA/MA+ caused an inward current but an increase (rather than a decrease) in pH(i). MA/MA+ did not cause any changes in H2O-injected oocytes. The MA/MA+-induced current and pH(i) increase were saturated at higher concentrations of MA/MA+. Amiloride inhibited MA/MA+-induced current and the increase in pH(i) in oocytes expressing Rhbg but had no effect on control oocytes. These results indicate that MA/MA+ is transported by Rhbg but differently than NH3/NH4+. The protonated MA+ is likely a direct substrate whose transport resembles that of NH4+. Transport of electroneutral MA is also enhanced by expression of Rhbg.

CFTR is required for PKA-regulated ATP sensitivity of Kir1.1 potassium channels in mouse kidney.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel plays vital roles in fluid transport in many epithelia. While CFTR is expressed along the entire nephron, its function in renal tubule epithelial cells remains unclear, as no specific renal phenotype has been identified in cystic fibrosis. CFTR has been proposed as a regulator of the 30 pS, ATP-sensitive renal K channel (Kir1.1, also known as renal outer medullar K [ROMK]) that is critical for K secretion by cells of the thick ascending limb (TAL) and distal nephron segments responsive to aldosterone. We report here that both ATP and glibenclamide sensitivities of the 30 pS K channel in TAL cells were absent in mice lacking CFTR and in mice homozygous for the deltaF508 mutation. Curcumin treatment in deltaF508-CFTR mice partially reversed the defect in ATP sensitivity. We demonstrate that the effect of CFTR on ATP sensitivity was abrogated by increasing PKA activity. We propose that CFTR regulates the renal K secretory channel by providing a PKA-regulated functional switch that determines the distribution of open and ATP-inhibited K channels in apical membranes. We discuss the potential physiological role of this functional switch in renal K handling during water diuresis and the relevance to renal K homeostasis in cystic fibrosis.

Characteristics of renal Rhbg as an NH4(+) transporter.

Rhbg is one of two recently cloned nonerythroid glycoproteins belonging to the Rh antigen family. Rhbg is expressed in basolateral membranes of intercalated cells of the kidney cortical collecting duct and some other cell types of the distal nephron and may function as NH(4)(+) transporters. The aim of this study was to characterize the role of Rhbg in transporting NH(4)(+). To do so, we expressed Rhbg in Xenopus laevis oocytes. Two-electrode voltage-clamp and H(+)-selective microlectrodes were used to measure NH(4)(+) currents, current-voltage plots, and intracellular pH (pH(i)). In oocytes expressing Rhbg, 5 mM NH(4)(+) induced an inward current of 93 +/- 7.7 nA (n = 20) that was significantly larger than that in control oocytes of -29 +/- 7.1 nA (P < 0.005). Whole cell conductance, at all tested potentials (-60 to +60 mV), was significantly more in oocytes expressing Rhbg compared with H(2)O-injected oocytes. In Rhbg oocytes, 5 mM NH(4)(+) depolarized the oocyte by 28 +/- 3.6 mV and decreased pH(i) by 0.30 +/- 0.04 at a rate of -20 +/- 2.5 x 10(-4) pH/s. In control oocytes, 5 mM NH(4)(+) depolarized V(m) by only 20 +/- 5.8 mV and pH(i) decreased by 0.07 +/- 0.01 at a rate of -2.7 +/- 0.6 x 10(-4) pH/s. Raising bath [NH(4)(+)] in increments from 1 to 20 mM elicited a proportionally larger decrease in pH(i) (DeltapH(i)), larger depolarization (DeltaV(m)), and a faster rate of pH(i) decrease. Bathing Rhbg oocytes in 20 mM NH(4)(+) induced an inward current of 140 +/- 7 nA that was not significantly different from 178 +/- 23 nA induced in H(2)O-injected (control) oocytes. The rate of pH(i) decrease induced by increasing external [NH(4)(+)] was significantly faster in Rhbg than in H(2)O-injected oocytes at all external NH(4)(+) concentrations. In oocytes expressing Rhbg, net NH(4)(+) influx (estimated from NH(4)(+)-induced H(+) influx) as a function of external [NH(4)(+)] saturated at higher [NH(4)(+)] with a V(max) of approximately 30.8 and an apparent K(m) of 2.3 mM (R(2) = 0.99). These data strongly suggest that Rhbg is a specific electrogenic transporter of NH(4)(+).

Requirement of voltage-gated calcium channel beta4 subunit for T lymphocyte functions.

Calcium is known to play vital roles in diverse physiological processes, and it is known that voltage-gated calcium channels (Cav) mediate calcium influx in excitable cells. However, no consensus exists on the molecular identity of the calcium channels present in nonexcitable cells such as T lymphocytes. Here, we demonstrate that T lymphocytes express both regulatory beta4 and poreforming Cav1 alpha1 subunits of Cav channels. Cav beta4-mutant T lymphocytes fail to acquire normal functions and display impairment in the calcium response, activation of the transcription factor NFAT, and cytokine production. Although Cav1 channels of lymphocytes retain their voltage dependency, T cell receptor stimulation dramatically increases channel opening, providing a new mechanism for calcium entry in lymphocytes.

Calcium-dependent, swelling-activated K+ conductance in human neuroblastoma cells.

In most mammalian cells, regulatory volume decrease (RVD) is mediated by swelling-activated Cl(-) and K(+) channels. Previous studies in the human neuroblastoma cell line CHP-100 have demonstrated that exposure to hypoosmotic solutions activates Cl(-) channels which are sensitive to Ca(2+). Whether a Ca(2+)-dependent K(+) conductance is activated after cell swelling was investigated in the present studies. Reducing the extracellular osmolarity from 290 to 190 mOsm/kg H(2)O rapidly activated 86Rb effluxes. Hypoosmotic stress also increased cytosolic Ca(2+) in fura-2 loaded cells. Pretreatment with 2.5 mM EGTA and nominally Ca(2+) free extracellular solution significantly decreased the hypoosmotically induced rise in cytosolic Ca(2+) and the swelling-activated 86Rb efflux. In cell-attached patch-clamp studies, decreasing the extracellular osmolarity activated a K(+) conductance that was blocked by Ba(2+). In addition, the swelling-activated K(+) channels were significantly inhibited in the presence of nominally free extracellular Ca(2+) and 2.5mM EGTA. These results suggest that in response to hypoosmotic stress, a Ca(2+)-dependent K(+) conductance is activated in the human neuroblastoma cell line CHP-100.

Active K+ secretion through multiple KCa-type channels and regulation by IKCa channels in rat proximal colon.

Colonic K+ secretion stimulated by cholinergic agents requires activation of muscarinic receptors and the release of intracellular Ca2+. However, the precise mechanisms by which this rise in Ca2+ leads to K+ efflux across the apical membrane are poorly understood. In the present study, Northern blot analysis of rat proximal colon revealed the presence of transcripts encoding rSK2 [small conductance (SK)], rSK4 [intermediate conductance (IK)], and rSlo [large conductance (BK)] Ca2+-activated K+ channels. In dietary K+-depleted animals, only rSK4 mRNA was reduced in the colon. On the basis of this observation, a cDNA encoding the K+ channel rSK4 was cloned from a rat colonic cDNA library. Transfection of this cDNA into Chinese hamster ovary (CHO) cells led to the expression of Ca2+-activated K+ channels that were blocked by the IK channel inhibitor clotrimazole (CLT). Confocal immunofluorescence confirmed the presence of IK channels in proximal colonic crypts, and Western blotting demonstrated that IK protein sorted to both the apical and basolateral surfaces of colonic epithelia. In addition, transcellular active K+ secretion was studied on epithelial strips of rat proximal colon using unidirectional 86Rb+ fluxes. The addition of thapsigargin or carbachol to the serosal surface enhanced net 86Rb+ secretion. The mucosal addition of CLT completely inhibited carbachol-induced net 86Rb+ secretion. In contrast, only partial inhibition was observed with the BK and SK channel inhibitors, iberiotoxin and apamin, respectively. Finally, in parallel with the reduction in SK4 message observed in animals deprived of dietary K+, carbachol-induced 86Rb+ secretion was abolished in dietary K+-depleted animals. These results suggest that the rSK4 channel mediates K+ secretion induced by muscarinic agonists in the rat proximal colon and that transcription of the rSK4 channel is downregulated to prevent K+ loss during dietary K+ depletion.

Paracellular Cl- permeability is regulated by WNK4 kinase: insight into normal physiology and hypertension.

Paracellular ion flux across epithelia occurs through selective and regulated pores in tight junctions; this process is poorly understood. Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension and hyperkalemia. Whereas WNK4 is known to regulate several transcellular transporters and channels involved in NaCl and K+ homeostasis, its localization to tight junctions suggests it might also regulate paracellular flux. We performed electrophysiology on mammalian kidney epithelia with inducible expression of various WNK4 constructs. Induction of wild-type WNK4 reduced transepithelial resistance by increasing absolute chloride permeability. PHAII-mutant WNK4 produced markedly larger effects, whereas kinase-mutant WNK4 had no effect. The electrochemical and pharmacologic properties of these effects indicate they are attributable to the paracellular pathway. The effects of WNK4 persist when induction is delayed until after tight-junction formation, demonstrating a dynamic effect. WNK4 did not alter the flux of uncharged solutes, or the expression or localization of selected tight-junction proteins. Transmission and freeze-fracture electron microscopy showed no effect of WNK4 on tight-junction structure. These findings implicate WNK signaling in the coordination of transcellular and paracellular flux to achieve NaCl and K+ homeostasis, explain PHAII pathophysiology, and suggest that modifiers of WNK signaling may be potent antihypertensive agents.