Gordon J. Cooper

Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane

We report here the application of a previously described method to directly determine the CO2 permeability (PCO2) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1‐expressing Xenopus laevis oocytes. This method measures the exchange of 18O between CO2, HCO3–, and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pHS) transients caused by the dominant effect of entry of CO2 vs. HCO3– into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces PCO2 from the normal value of 0.15 ± 0.08 (SD; n85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases PCO2 2‐fold and doubles the alkaline pHS gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces PCO2 of RBCs solely by action on AQP1 as it has no effect in AQP1‐deficient RBCs. 3) PCO2 determinations of RBCs and pHS measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS‐sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high PCO2 of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total PCO2.—Endeward, V., Musa‐Aziz, R., Cooper, G. J., Chen, L., Pelletier, M. F., Virkki, L. V., Supuran, C. T., King, L. S., Boron, W. F., Gros, G. Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane. FASEB J. 20, 1974–1981 (2006)

Transport of volatile solutes through AQP1

For almost a century it was generally assumed that the lipid phases of all biological membranes are freely permeable to gases. However, recent observations challenge this dogma. The apical membranes of epithelial cells exposed to hostile environments, such as gastric glands, have no demonstrable permeability to the gases CO2 and NH3. Additionally, the water channel protein aquaporin 1 (AQP1), expressed at high levels in erythrocytes, can increase membrane CO2 permeability when expressed in Xenopus oocytes. Similarly, nodulin‐26, which is closely related to AQP1, can act as a conduit for NH3. A key question is whether aquaporins, which are abundant in virtually every tissue that transports O2 and CO2 at high levels, ever play a physiologically significant role in the transport of small volatile molecules. Preliminary data are consistent with the hypothesis that AQP1 enhances the reabsorption of HCO3− by the renal proximal tubule by increasing the CO2 permeability of the apical membrane. Other preliminary data on Xenopus oocytes heterologously expressing the electrogenic Na+‐HCO3− cotransporter (NBC), AQP1 and carbonic anhydrases are consistent with the hypothesis that the macroscopic cotransport of Na+ plus two HCO3− occurs as NBC transports Na+ plus CO32‐ and AQP1 transports CO2 and H2O. Although data ‐ obtained on AQP1 reconstituted into liposomes or on materials from AQP1 knockout mice ‐ appear inconsistent with the model that AQP1 mediates substantial CO2 transport in certain preparations, the existence of unstirred layers or perfusion‐limited conditions may have masked the contribution of AQP1 to CO2 permeability.

Expression of a sodium bicarbonate cotransporter in human parotid salivary glands

The human parotid gland secretes much of the bicarbonate that enters the mouth. Prompted by studies of animal models, this study sought evidence for the expression of a functional Na(+)-HCO(3)(-) cotransporter (NBC) in human parotid acinar cells. Microfluorometric measurements of intracellular pH in isolated acini showed that the recovery from an acid load was achieved in part by HCO(3)(-) uptake via a Na(+)-dependent, DIDS-sensitive mechanism. By reverse transcriptase-polymerase chain reaction, a full-length NBC1 clone was obtained showing more than 99% homology with the human pancreatic isoform hpNBC1. Expressed in Xenopus oocytes, the electrogenicity of the transporter was detected as an inwardly directed, Na(+)- and HCO(3)(-)-dependent flux of negative charge. Immunohistochemistry using antibodies raised to NBC1 showed strong staining of the basolateral membrane of the acinar cells. Therefore, it was concluded that a functional electrogenic Na(+)-HCO(3)(-) cotransporter is expressed in the human parotid gland, and that it contributes to pH regulation in the acinar cells and could play a significant part in salivary secretion.

Functional and developmental expression of a zebrafish Kir1.1 (ROMK) potassium channel homologue Kcnj1

Non‐technical summary  Due to the conservation of developmental pathways and genetic material over the course of evolution, non‐mammalian ‘model organisms’ such as the zebrafish embryo are emerging as valuable tools to explore causes and potential treatments for human diseases. Ion channels are proteins that form pores and help to establish and control electrical gradients by allowing the flow of ions across biological membranes. A diverse range of key physiological mechanisms in every organ in the body depends on the activity of ion channels. In this paper, we show that a potassium‐selective channel that underlies salt reabsorption and potassium excretion in the human kidney is also expressed in zebrafish in cells that are important regulators of salt balance. Disruption of the channels expression in zebrafish leads to effects on the activity of the heart, consistent with a role for this channel in the control of potassium balance in the embryo.

Vasopressin regulation of the renal UT-A3 urea transporter

Facilitative urea transporters in the mammalian kidney play a vital role in the urinary concentrating mechanism. The urea transporters located in the renal inner medullary collecting duct, namely UT-A1 and UT-A3, are acutely regulated by the antidiuretic hormone vasopressin. In this study, we investigated the vasopressin regulation of the basolateral urea transporter UT-A3 using an MDCK-mUT-A3 cell line. Within 10 min, vasopressin stimulates urea flux through UT-A3 transporters already present at the plasma membrane, via a PKA-dependent process. Within 1 h, vasopressin significantly increases UT-A3 localization at the basolateral membrane, causing a further increase in urea transport. While the basic trafficking of UT-A3 to basolateral membranes involves both protein kinase C and calmodulin, its regulation by vasopressin specifically occurs through a casein kinase II-dependent pathway. In conclusion, this study details the effects of vasopressin on UT-A3 urea transporter function and hence its role in regulating urea permeability within the renal inner medullary collecting duct.

Alpha‐aminoisobutyric acid transport in rat soleus muscle and its modification by membrane stabilizers and insulin.

The non‐metabolizable amino acid alpha‐aminoisobutyric acid (AIB) has been used to study the effects of insulin and a number of membrane stabilizers on amino acid transport in rat soleus muscle in an attempt to characterize the mechanisms present. 2. Insulin (5‐‐100 millimicron./ml) increases the net uptake of AIB two‐ to threefold. Since insulin is without significant effects on AIB efflux, stimulation of net uptake appears to result directly from an increased AIB influx. 3. All classes of membrane stabilizer tested affected AIB fluxes but the responses observed varied for different classes of compounds. 4. The total anaesthetic, tetracaine, reduced AIB accumulation both in the absence and presence of insulin by a similar proportion. The effects on AIB efflux were dependent on the concentration of tetracaine used. Efflux was suppressed by concentrations up to 1 mM whereas 4 mM‐tetracaine caused a massive stimulation of AIB efflux. Other local anaesthetics and barbiturates produced similar effects. 5. Another group of membrane stabilizers, exemplified by chlorpromazine, also suppressed AIB uptake, but at no concentration did they reduce AIB efflux. In fact, efflux of AIB began to be increased at concentrations of chlorpromazine which were giving only a modest inhibition of uptake. 6. Measurement of the initial rate of uptake showed that it involved two components. Tetracaine appears to inhibit the saturable component whilst leaving the non‐saturable component relatively unaffected. 7. The active uptake of AIB was shown to be Na+‐dependent, and under Na+‐free conditions tetracaine had no effect on the initial rate of uptake. The non‐saturable component was also shown to be Na+‐sensitive, uptake from high extracellular concentrations of AIB being reduced in Na+‐free media. 8. The possibility of the presence of a carrier‐mediated AIB efflux mechanism was investigated. AIB efflux was stimulated by extracellular AIB (homo‐exchange) or glycine (hetero‐exchange), but not mannitol. This suggests the involvement of a carrier‐mediated process in AIB efflux. 9. The present study has demonstrated a heterogeneity among different classes of membrane stabilizers in their actions on AIB efflux which is in marked contrast to previous observations of sugar and cation transport in this preparation. Possible reasons for these differences are discussed.

Adaptive downregulation of a quinidine-sensitive cation conductance in renal principal cells of TWIK-1 knockout mice

TWIK-1, a member of the two-pore domain K+ channel family, is expressed in brain, kidney, and lung. The aim of this study was to examine the effect of loss of TWIK-1 on the renal cortical collecting duct. Ducts were isolated from wild-type and TWIK-1 knockout mice by enzyme digestion and whole-cell clamp obtained via the basolateral membrane. Current- and voltage-clamp approaches were used to examine K+ conductances. No difference was observed between intercalated cells from wild-type or knockout ducts. In contrast, knockout principal cells were hyperpolarized compared to wild-type cells and had a reduced membrane conductance. This was a consequence of a fall in a barium-insensitive, quinidine-sensitive conductance (GQuin). GQuin demonstrated outward rectification and had a relatively low K+ to Na+ selectivity ratio. Loss of GQuin would be expected to lead to the hyperpolarization observed in knockout ducts by increasing fractional K+ conductance and Na+ uptake by the cell. Consistent with this hypothesis, knockout ducts had an increased diameter in comparison to wild-type ducts. These data suggest that GQuin contributes to the resting membrane potential in the cortical collecting duct and that a fall in GQuin could be an adaptive response in TWIK-1 knockout ducts.

A Kir2.3-like K + Conductance in Mouse Cortical Collecting Duct Principal Cells

K+ channels play an important role in renal collecting duct cell function. The current study examined barium (Ba2+)-sensitive whole-cell K+ currents (IKBa) in mouse isolated collecting duct principal cells. IKBa demonstrated strong inward rectification and was inhibited by Ba2+ in a dose- and voltage-dependent fashion, with the Kd decreasing with hyperpolarization. The electrical distance of block by Ba2+ was around 8.5%. As expected for voltage-dependent inhibition, the association constant increased with hyperpolarization, suggesting that the rate of Ba2+ entry was increased at negative potentials. The dissociation constant also increased with hyperpolarization, consistent with the movement of Ba2+ ions into the intracellular compartment at negative potentials. These properties are not consistent with ROMK but are consistent with the properties of Kir2.3. Kir2.3 is thought to be the dominant basolateral K+ channel in principal cells. This study provides functional evidence for the expression of Kir2.3 in mouse cortical collecting ducts and confirms the expression of Kir2.3 in this segment of the renal tubule using reverse-transcriptase polymerase chain reaction. The conductance described here is the first report of a macroscopic K+ conductance in mouse principal cells that shares the biophysical profile of Kir2.3. The properties and dominant nature of the conductance suggest that it plays an important role in K+ handling in the principal cells of the cortical collecting duct.

Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions.

Abstract. Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K+ and HCO−3 out of the cell, tending to lower intracellular pH (pHi). With an inwardly directed K/HCO3 gradient, the cotransporter mediates a net uptake of alkali (i.e., K+ and HCO−3 influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA+) inhibit the inwardly directed cotransporter by interacting at the intracellular K+ site. We computed the equivalent HCO−3 influx (JHCO3) mediated by the cotransporter from the rate of pHi increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pHi 8.0, using a dialysis fluid (DF) free of K+, Na+ and Cl−. Our standard artificial seawater (ASW) also lacked Na+, K+ and Cl−. After halting dialysis, we introduced an ASW containing 437 mm K+ and 0.5% CO2/12 mm HCO−3, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO2 influx, followed by a slower plateau-phase pHi increase, due to inward cotransport of K+ and HCO−3. With no QA+ in the DF, JHCO3 was ∼58 pmole cm−2 sec−1. With 400 mm tetraethylammonium (TEA+) in the DF, JHCO3 was virtually zero. The apparent Ki for intracellular TEA+ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K+ channels. Introducing 100 mm inhibitor into the DF reduced JHCO3 to ∼20 pmole cm−2 sec−1 for tetramethylammonium (TMA+), ∼24 for TEA+, ∼10 for tetrapropylammonium (TPA+), and virtually zero for tetrabutylammonium (TBA+). The apparent Ki value for TBA+ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA+), with an apparent Ki of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO3 influx in the potency sequence PPTEA+ > TBA+ > TPA+ > TEA+≅ TMA+. The identification of inhibitors of the K/HCO3 cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.

CrossTalk proposal: Physiological CO2 exchange can depend on membrane channels

Since the discovery that CO2 passes through aquaporin-1 (AQP1; Nakhoul et al. 1998; Cooper & Boron, 1998), the importance of channelvs. lipid-mediated gas transport has often been portrayed as an either/or issue. However, depending upon physiological context, the role of channels may be insignificant or dominant. In a landmark study, Mitchell (1830) examined gas permeation across barriers of natural rubber or animal tissue, rank-ordered the ‘relative facility of transmission’ of several gases, and recognized that these move independently of one another in a mechanism dependent upon ‘infiltration’ (i.e. solubility) in the organic molecular barrier – the first statement of ‘solubility theory’. Later, Graham showed that permeation across rubber membranes depends on not only solubility but also diffusion through the barrier (Graham, 1866) – the first statement of ‘solubility–diffusion theory’. Meanwhile, Fick proposed his law of mass diffusion, which Wroblewski combined with Henry’s