Emile L. Boulpaep

An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel

Two electroneutral, Na+-driven HCO-3 transporters, the Na+-driven Cl-/HCO-3 exchanger and the electroneutral Na+/ HCO-3 cotransporter, have crucial roles in regulating intracellular pH in a variety of cells, including cardiac myocytes, vascular smooth-muscle, neurons and fibroblasts; however, it is difficult to distinguish their Cl- dependence in mammalian cells. Here we report the cloning of three variants of an electroneutral Na+/HCO-3 cotransporter, NBCn1, from rat smooth muscle. They are 89–92% identical to a human skeletal muscle clone, 55–57% identical to the electrogenic NBCs and 33–43% identical to the anion exchangers. When expressed in Xenopus oocytes, NBCn1-B (which encodes 1,218 amino acids) is electroneutral, Na+-dependent and HCO-3-dependent, but not Cl--dependent. Oocytes injected with low levels of NBCn1-B complementary RNA exhibit a Na+ conductance that 4,4-diisothiocyanatostilbene-2,2′-disulphonate stimulates slowly and irreversibly.

Immunolocalization of the electrogenic Na+-HCO3/- cotransporter in mammalian and amphibian kidney

Electrogenic cotransport of Na+ and[Formula: see text] is a crucial element of[Formula: see text] reabsorption in the renal proximal tubule (PT). An electrogenic Na+-[Formula: see text]cotransporter (NBC) has recently been cloned from salamander and rat kidney. In the present study, we generated polyclonal antibodies (pAbs) to NBC and used them to characterize NBC on the protein level by immunochemical methods. We generated pAbs in guinea pigs and rabbits by immunizing with a fusion protein containing the carboxy-terminal 108 amino acids (amino acids 928-1035) of rat kidney NBC (rkNBC). By indirect immunofluorescence microscopy, the pAbs strongly labeled HEK-293 cells transiently expressing NBC, but not in untransfected cells. By immunoblotting, the pAbs recognized a ∼130-kDa band in Xenopus laevis oocytes expressing rkNBC, but not in control oocytes injected with water or cRNA for the Cl-/[Formula: see text]exchanger AE2. In immunoblotting experiments on renal microsomes, the pAbs specifically labeled a major band at ∼130 kDa in both rat and rabbit, as well as a single ∼160-kDa band in salamander kidney. By indirect immunofluorescence microscopy on 0.5-μm cryosections of rat and rabbit kidneys fixed in paraformaldehyde-lysine-periodate (PLP), the pAbs produced a strong and exclusively basolateral staining of the PT. In the salamander kidney, the pAbs labeled only weakly the basolateral membrane of the PT. In contrast, we observed strong basolateral labeling in the late distal tubule, but not in the early distal tubule. The specificity of the pAbs for both immunoblotting and immunohistochemistry was confirmed in antibody preabsorption experiments using either the fusion protein used for immunization or similarly prepared control fusion proteins. In summary, we have developed antibodies specific for NBC, determined the apparent molecular weights of rat, rabbit, and salamander kidney NBC proteins, and described the localization of NBC within the kidney of these mammalian and amphibian species.

Chloride transport across the basolateral cell membrane of theNecturus proximal tubule: Dependence on bicarbonate and sodium

SummaryThe transport of chloride across theNecturus proximal tubule cell was studied in the doubly-perfused kidney using conventional, chloride-sensitive and pH-sensitive microelectrodes. Lowering chloride activity in the basolateral solution results in a reduction in intracellular Cl− activity (aCli). This reduction inaCli is inhibited by removing either HCO3− or Na+ from the perfusion solution, indicating that both HCO3− and Na+ are required for Cl− movement across the basolateral cell membrane. Reducing either HCO3− or Na+ in the basolateral solution causes an increase inaCli. Thus changes in either Na+ or HCO3− chemical gradients across the basolateral cell membrane significantly affect chloride movement. Changing intracellular pH by means of NH4Cl exposure results in an increase inaCli followed by a sharp decrease when NH4Cl is removed. These changes in intracellular chloride do not occur in the absence of HCO3−. Likewise, the decrease inaCli following NH4Cl treatment requires the presence of Na+ in the basolateral solution. We conclude that chloride is transported across the basolateral cell membrane in exchange for both Na+ and HCO3−. Our results also support the presence of a Na+/Cl− contransport mechanism on the apical cell membrane.

Effect of amiloride on the apical cell membrane cation channels of a sodium-absorbing, potassium-secreting renal epithelium.

SummaryThe effect of the K-sparing diuretic amiloride was assessed electrophysiologically in the isolated cortical collecting tubule of the rabbit, a segment which absorbs Na and secretes K. Low concentrations of amiloride in the perfusate caused a rapid, reversible, decrease in the magnitude of the lumen negative transepithelial potential difference,Vte, transepithelial conductanceGte, and equivalent short-circuit current,Isc, with an apparentK1/2 of approximately 7×10−8m. The effects of a maximum inhibitory concentration of amiloride (10−5m) were identical to those observed upon Na removal from lumen and bath (Na removal from the bath alone has no effect). Removal of Na in the presence of 10−5m amiloride had no affect onVte,Gte, orIsc, and is consistent with the view that amiloride blocks the Na conductive pathways of the apical cell membrane. Further, in the absence of Na, the subsequent addition of amiloride had no influence. In tubules where active Na absorption was either spontaneously low, or abolished by removal of Na from lumen and bath, the elevation of K from 5 to 155 meq/liter in the perfusate caused a marked change of theVte in the negative direction and an increase in theGte. These effects could be attributed to a high K permeability of the apical cell membrane and not of the tight junctions. Amiloride (10−5m) had no effect on these responses to K. It is concluded that amiloride selectively blocks the apical cell membrane Na channels but has no effect on the K conductive pathways(s). This selective nature of amiloride may indicate that Na and K are transported across the apical cell membrane via separate conductive pathways.

Regulation of volume‐activated chloride channels by P‐glycoprotein: phosphorylation has the final say!

1 P‐glycoprotein (Pgp) is a transmembrane transporter causing efflux of a number of chemically unrelated drugs and is responsible for resistance to a variety of anticancer drugs during chemotherapy. 2 Pgp overexpression in cells is also associated with volume‐activated chloride channel activity; Pgp is thought to regulate such activity. 3 Reversible phosphorylation is a possible mechanism for regulating the transport and chloride channel regulation functions of Pgp. Protein kinase C (PKC) is a good candidate for inducing such phosphorylation. 4 Hierarchical multiple phosphorylation (e.g. of different serines and with different PKC isoforms) may shuttle the protein between its different states of activity (transport or channel regulation). Cell volume changes may trigger phosphorylation of Pgp at sites causing inhibition of transport. 5 The possible regulation of chloride channels by Pgp and the potential involvement of reversible phosphorylation in such regulation is reviewed.

Pressure Control of Sodium Reabsorption and Intercellular Backflux across Proximal Kidney Tubule

The magnitude of changes in luminal hydrostatic pressure (DeltaP(L)), peritubular capillary hydrostatic pressure (DeltaP(PT)), and peritubular capillary colloid osmotic pressure (Deltapi) was determined in the Necturus kidney during volume expansion (VE). The specific effects of separate changes of each pressure parameter on proximal net sodium transport (J(Na)) were studied in isolated perfused kidneys. The combined effect of DeltaP(L), DeltaP(PT), and Deltapi, of a magnitude similar to that induced by volume expansion, decreases J(Na) by 26% in the perfused kidney. A major portion of the natriuresis in VE is due to changes in intrarenal pressures. The effect of Deltapi on the permeability characteristics of Necturus proximal tubule was studied. With increasing Deltapi, the ionic conductance of the paracellular shunt pathway decreased, since transepithelial input and specific resistance rose significantly, whereas cellular membrane resistance remained unchanged. Transepithelial permeability coefficients for sodium chloride and raffinose changed inversely proportional to transepithelial resistance, indicating an alteration of a paracellular permeation route. Net passive sodium backflux and active transport flux components were calculated. Increased net sodium transport with rising Deltapi is accompanied by a significant drop in passive back diffusion, without an increment in the active flux component. Change in passive sodium ion back diffusion thus appears to be a key physiological factor in the control of transepithelial sodium transport.

Molecular cloning of a glibenclamide-sensitive, voltage-gated potassium channel expressed in rabbit kidney.

Shaker genes encode voltage-gated potassium channels (Kv). We have shown previously that genes from Shaker subfamilies Kv1.1, 1.2, 1.4 are expressed in rabbit kidney. Recent functional and molecular evidence indicate that the predominant potassium conductance of the kidney medullary cell line GRB-PAP1 is composed of Shaker-like potassium channels. We now report the molecular cloning and functional expression of a new Shaker-related voltage-gated potassium channel, rabKv1.3, that is expressed in rabbit brain and kidney medulla. The protein, predicted to be 513 amino acids long, is most closely related to the Kv1.3 family although it differs significantly from other members of that family at the amino terminus. In Xenopus oocytes, rabKv1.3 cRNA expresses a voltage activated K current with kinetic characteristics similar to other members of the Kv1.3 family. However, unlike previously described Shaker channels, it is sensitive to glibenclamide and its single channel conductance saturates. This is the first report of the functional expression of a voltage-gated K channel clone expressed in kidney. We conclude that rabKv1.3 is a novel member of the Shaker superfamily that may play an important role in renal potassium transport.

Electrical properties of chloride transport across the Necturus proximal tubule

SummaryThe chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl−. When extracellular Cl− activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl− conductance of the paracellular shunt but a low Cl− conductance of the cell membranes. Reduction of Cl− in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl− perfusion was abolished by exposure to HCO3−-free solutions and SITS treatment. In control conditions, intracellular Cl− activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl− in only the basolateral solution caused a decrease in intracellular Cl−. From an estimate of the net Cl− flux across the basolateral cell membrane and the electrochemical driving force, a Cl− conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl− conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl− by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl− transport across the basolateral cell membrane of theNecturus proximal tubule cell.

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.

A calcium-activated and nucleotide-sensitive nonselective cation channel in M-1 mouse cortical collecting duct cells.

We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA89:10262–10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na+, K+, Rb+, Cs+, and Li+. It has a linear I-V relation with a conductance of 22.7±0.5 pS (n=78) at room temperature. The Pcation/ Panion ratio is about 60 and there is no measurable conductance for NMDG, Ca2+, Ba2+, and Mn2+. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10−6m and depolarization increases channel activity (NPo). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10−4m and 10−3m, ATP reduces NPo by 23% and 69%, respectively.Furthermore, since ADP (10−3m) reduces NPo by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a γ-phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10−4m) or by cGMP-dependent protein kinase (10−7m) in the presence of 8-Br-cGMP (10−5m) and ATP (10−4m). The NSC channel is not sensitive to amiloride (10−4m cytoplasmic and/or extracellular) but flufenamic acid (10−4m) produces a voltage-dependent block, reducing NPo by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages.We conclude that the NSC channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.