Nanna Macaulay - Academia.edu (original) (raw)

Papers by Nanna Macaulay

Neurochemical Research, 2012

An important feature of neuronal signalling is the increased concentration of K(+) in the extrace... more An important feature of neuronal signalling is the increased concentration of K(+) in the extracellular space. The K(+) concentration is restored to its original basal level primarily by uptake into nearby glial cells. The molecular mechanisms by which K(+) is transferred from the extracellular space into the glial cell are debated. Although spatial buffer currents may occur, their quantitative contribution to K(+) clearance is uncertain. The concept of spatial buffering of K(+) precludes intracellular K(+) accumulation and is therefore (i) difficult to reconcile with the K(+) accumulation repeatedly observed in glial cells during K(+) clearance and (ii) incompatible with K(+)-dependent glial cell swelling. K(+) uptake into non-voltage clamped cultured glial cells is carried out by the Na(+)/K(+)-ATPase and the Na(+)/K(+)/Cl(-) cotransporter in combination. In brain slices and intact optic nerve, however, only the Na(+)/K(+)-ATPase has been demonstrated to be involved in stimulus-evoked K(+) clearance. The glial cell swelling associated with K(+) clearance is prevented under conditions that block the activity of the Na(+)/K(+)/Cl(-) cotransporter. The Na(+)/K(+)/Cl(-) cotransporter is activated by increased K(+) concentration and cotransports water along with its substrates. It thereby serves as a K(+)-dependent molecular water pump under conditions of increased extracellular K(+) load.

Gap junction channels are composed of two hemichannels (connexons) from neighboring cells. Undock... more Gap junction channels are composed of two hemichannels (connexons) from neighboring cells. Undocked connexons have been proposed to form open channels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules with masses lower than 1 kDa. A range of physiologically relevant molecules, smaller than the assumed molecular cutoff of 1 kDa, have therefore been proposed to permeate connexin 43 (Cx43) in its hemichannel configuration. However, the permeability profile of Cx43 hemichannels remains unresolved. Exposure of Cx43-expressing Xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. In spite thereof, a range of smaller biological molecules, such as water, glutamate, lactate, and glucose, did not gain detectable access through the pore whereas ATP did. In contrast, permeability of glutamate, glucose and ATP was...

Gap-junctions are intracellular channels connecting the cytoplasms of a variety of mammalian cell... more Gap-junctions are intracellular channels connecting the cytoplasms of a variety of mammalian cells: Adjacent connexons/hemichannels from two opposing cells dock in the extracellular space to form the communicating gap junction channel. It has become increasingly evident that connexons appear unopposed in the plasma membrane, and that opening of these likely occur under pathophysiological conditions or experimental condition mimicking those. Due to its large-diameter central pore (15 Å; Unger et al. 1999), opening of hemichannels has been suggested leading to release of substances which may be harmful to the cell itself and/or surrounding cells. The study of connexin 43 (Cx43) hemichannels/connexons in native settings is challenged by the growing list of channels (such as P2X-receptors and pannexins) regulated by the same stimuli and cross-specificity of the inhibitors. To perform a more biophysical analysis of the permeability profile of Cx43 hemichannels, Cx43 was functionally expr...

Connexons form the Gap junction, connecting adjacent cells. Each Connexon is composed of six Conn... more Connexons form the Gap junction, connecting adjacent cells. Each Connexon is composed of six Connexin subunits (Cx), with Cx43 being the main connexin expressed in astrocytes. The Gap Junction is established when two adjacent Connexons, dock in the extracellular space, forming a pathway for cell to cell communication. Ions and several biologically active molecules such as ATP/ADP and IP3 pass between cells through Gap Junctions allowing cell to cell communication or synchronization. Additionally, Connexons appear as undocked Hemichannels, potentially forming a channel connecting the cytoplasm with the extracellular space. Hemichannel opening has been reported in vivo following exposure to a multitude of molecular clues, including metabolic inhibition, pH, pathophysiological ion concentrations or loss of membrane polarization; hallmark events of pathofysiological events such as ischemia or spreading depression. It has been speculated that opening of hemichannels allow passage of subs...

Wiley Interdisciplinary Reviews: Membrane Transport and Signaling, 2012

Water transport across cell membranes is central to most physiological functions. About 200 L of ... more Water transport across cell membranes is central to most physiological functions. About 200 L of water move across epithelial cells each day in humans in order to maintain whole-body homeostasis; water transport in and out of organs such as the brain and eye are of major clinical importance. It is well established that the water transport is driven by ion transport, but how? Osmosis is not always the answer: water can be transported against considerable osmotic gradients, apparently without any external osmotic or hydrostatic driving forces. It is generally accepted that cotransporters of the symport type play a key role for the coupling between ion and water fluxes. Models of coupling are either molecular or based on unstirred layer effects, and can be distinguished by their response time: for molecular models, water transport follows changes of substrate transport instantaneously; in unstirred layer models there is a delay while the osmolarity changes in the solutions surrounding the cotransport protein. For cotransporters expressed heterologously in Xenopus oocytes, influx of water can be detected about 1 second after initiation of cotransport of ions and other substrates. This is 20 times faster than expected (and observed) for unstirred layer effects. Water transport in cotransporters is best explained by a molecular model in which ion and water fluxes are coupled by a mechanism within the protein. This would also clarify how cotransporters exploit the free energy in the ion fluxes for the uphill transport of water.

Biochemical Society Transactions, 2015

Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardia... more Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardiac and brain function. Gap junctions are formed by the docking of two hemichannels from neighbouring cells. Undocked Cx43 hemichannels can upon different stimuli open towards the extracellular matrix and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current knowledge of protein kinase C (PKC)-dependent regulation of Cx43 and discuss the divergent results.

The Journal of physiology, 2012

The NKCC1 and NKCC2 isoforms of the mammalian Na⁺–K⁺–2Cl⁻ cotransporter were expressed in Xenopus... more The NKCC1 and NKCC2 isoforms of the mammalian Na⁺–K⁺–2Cl⁻ cotransporter were expressed in Xenopus oocytes and the relation between external ion concentration and water fluxes determined.Water fluxes were determined from changes in the oocytes volume and ion fluxes from 86Rb+ uptake. Isotonic increases in external K⁺ concentration elicited abrupt inward water fluxes in NKCC1; the K⁺ dependence obeyed one-site kinetics with a K₀.₅ of 7.5 mM. The water fluxes were blocked by bumetanide, had steep temperature dependence and could proceed uphill against an osmotic gradient of 20 mosmol l⁻¹. A comparison between ion and water fluxes indicates that 460 water molecules are cotransported for each turnover of the protein. In contrast, NKCC2 did not support water fluxes.Water transport in NKCC1 induced by increases in the external osmolarity had high activation energy and was blocked by bumetanide. The osmotic effects of NaCl were smaller than those of urea and mannitol. This supports the noti...

International review of cytology, 2002

Molecular water pumps are membrane proteins of the cotransport type in which a flux of water is c... more Molecular water pumps are membrane proteins of the cotransport type in which a flux of water is coupled to substrate fluxes by a mechanism within the protein. Free energy can be exchanged between the fluxes. Accordingly, the flux of water may be relatively independent of the external water chemical potential and can even proceed uphill. In short, water is being cotransported. The evidence for water cotransport is reviewed with particular emphasis on electrogenic cotransporters expressed in Xenopus oocytes under voltage clamped conditions. Phenomena such as uphill water transport, tight coupling between water transport and clamp current, cotransport of small hydrophilic molecules, and shifts in reversal potentials with osmolarity are discussed with examples from the Na+/glutamate and Na+/glucose cotransporters. Unstirred layers and electrode artifacts as alternative explanations for such cotransport can be ruled out for both experimental and theoretical reasons. Indeed, substrate flu...

International review of cytology, 2002

Three kinds of membrane proteins have been shown to have water channels properties: the aquaporin... more Three kinds of membrane proteins have been shown to have water channels properties: the aquaporins, the cotransporters, and the uniports. A molecular-kinetic description of water transport in pores is compared to analytical models based on macroscopic parameters such as pore diameter and length. The use and limitations of irreversible thermodynamics is discussed. Experimental data on water and solute permeability in aquaporins are reviewed. No unifying transport model based on macroscopic parameters can be set up; for example, there is no correlation between solute diameter and permeability. Instead, the influence of hydrogen bonds between solute and pore, and the pH dependence of permeability, point toward a model based upon chemical interactions. The atomic model for AQP1 based on electron crystallographic data defines the dimensions and chemical nature of the aqueous pore. These structural data combined with quantum mechanical modeling and computer simulation might result in a re...

Advances in Experimental Medicine and Biology, 2005

... Morten Grunnet, Thomas Jespersen, Nanna K. Jørgensen, Nanna MacAulay, Nicole Schmitt, Olaf Po... more ... Morten Grunnet, Thomas Jespersen, Nanna K. Jørgensen, Nanna MacAulay, Nicole Schmitt, Olaf Pongs, Henrik S. Jensen, Søren-Peter Olesen and Dan A. Klaerke* 1. ABSTRACT 1 ... Kenneth BE Gagnon, Peter K. Lauf, and Robert EW Fyffe* 1. INTRODUCTION ...

Journal of Biological Chemistry, 2014

The permeability and physiological role of several large-pore (hemi)channels are unresolved. Resu... more The permeability and physiological role of several large-pore (hemi)channels are unresolved. Results: Large-pore (hemi)channels, when heterologously expressed, display isoformspecific permeability and gating for ions and fluorescent dyes. Conclusion: Large-pore channels have isoformspecific transport characteristics that can be used for their identification. Significance: Although large-pore channels have characteristic properties in overexpression systems, these properties may be undetectable in native cells.

The Journal of Physiology, 2003

The Journal of Physiology, 2001

Cotransporters are membrane proteins in which the transmembrane fluxes of various substrates are ... more Cotransporters are membrane proteins in which the transmembrane fluxes of various substrates are coupled: the downhill flux of one substrate energizes the uphill flux of another. Evidence is accruing, however, that cotransporters, in addition to their recognized functions, also play a direct role in water transport. All cotransporters of the symport type tested so far exhibit cotransport of water, in which a fixed number of water molecules are translocated together with non-aqueous substrates by a process that is independent of external parameters. In addition, the cotransporters can function as passive water channels. The K + -Cl _ and H + -lactate cotransporters have been found to cotransport 500 water molecules per turnover . The human Na + -glucose cotransporter, the rabbit Na + -glucose cotransporter and the Na + -dicarboxylate cotransporter cotransport 210, 390 and 175 water molecules per turnover, respectively . Osmotic water transport has been described in detail for the Na + -glucose and the Na + -GABA transporters ).

The Journal of Physiology, 2013

We test a novel model of osmosis in aquaporins. • A solute molecule present at the pore mouth can... more We test a novel model of osmosis in aquaporins. • A solute molecule present at the pore mouth can be reflected or permeate the pore; we propose that only reflected molecules induce osmotic water transport, while permeating molecules give rise to no water transport. • We tested a range of channel geometries using aquaporins AQP1 and AQP9 and mutants thereof; the aquaporins were expressed in Xenopus oocytes. Osmotic gradients were generated by solutes of molecular weights in the range 45-595 Daltons. The reflected fraction of a given solute was estimated optically and compared to the permeability obtained from uptakes of radio-labelled solutes. • In accordance with our model there was a linear relationship between solute permeability and reflection coefficient: solutes with high permeability had low reflection coefficients and vice versa. • We found no evidence for significant coupling between water and solute fluxes inside the pore. Abstract We test a novel, stochastic model of osmotic water transport in aquaporins. A solute molecule present at the pore mouth can either be reflected or permeate the pore. We assume that only reflected solute molecules induce osmotic transport of water through the pore, while permeating solute molecules give rise to no water transport. Accordingly, the rate of water transport is proportional to the reflection coefficient σ, while the solute permeability, P S , is proportional to 1 -σ. The model was tested in aquaporins heterologously expressed in Xenopus oocytes. A variety of aquaporin channel sizes and geometries were obtained with the two aquaporins AQP1 and AQP9 and mutant versions of these. Osmotic water transport was generated by adding 20 mM of a range of different-sized osmolytes to the outer solution. The osmotic water permeability and the reflection coefficient were measured optically at high resolution and compared to the solute permeability obtained from short-term uptake of radio-labelled solute under isotonic conditions. For each type of aquaporin there was a linear relationship between solute permeability and reflection coefficient, in accordance with the model. We found no evidence for coupling between water and solute fluxes in the pore. In confirmation of molecular dynamic simulations, we conclude that the magnitude of the osmotic water permeability and the reflection coefficient are determined by processes at the arginine selectivity filter located at the outward-facing end of the pore.

The Journal of Physiology, 2002

The Journal of Physiology, 2002

The Journal of Physiology, 2006

The glucose transporter GLUT2 has been shown to also transport water. In the present paper we inv... more The glucose transporter GLUT2 has been shown to also transport water. In the present paper we investigated the relation between sugar and water transport in human GLUT2 expressed in Xenopus oocytes. Sugar transport was determined from uptakes of non-metabolizable glucose analogues, primarily 3-O-methyl-D-glucopyranoside; key experimental results were confirmed using D(+)-glucose. Water transport was derived from changes in oocyte volume monitored at a high resolution (20 pl, 1 s). Expression of GLUT2 induced a sugar permeability, P S , of about 5 × 10 −6 cm s −1 and a passive water permeability, L p , of 5.5 × 10 −5 cm s −1 . Accordingly, the passive water permeability of a GLUT2 protein is about 10 times higher than its sugar permeability. Both permeabilities were abolished by phloretin. Isosmotic addition of sugar to the bathing solution (replacing mannitol) induced two parallel components of water influx in GLUT2, one by osmosis and one by cotransport. The osmotic driving force arose from sugar accumulation at the intracellular side of the membrane and was given by an intracellular diffusion coefficient for sugar of 10 −6 cm 2 s −1 , one-fifth of the free solution value. The diffusion coefficient was determined in oocytes coexpressing GLUT2 and the water channel AQP1 where water transport was predominantly osmotic. By the cotransport mechanism about 35 water molecules were transported for each sugar molecule by a mechanism within the GLUT2. These water molecules could be transported uphill, against an osmotic gradient, energized by the flux of sugar. This capacity for cotransport is 10 times smaller than that of the Na + -coupled glucose transporters (SGLT1). The physiological role of GLUT2 for intestinal transport under conditions of high luminal sugar concentrations is discussed.

Pflügers Archiv, 2002

Ca 2+ -activated K + channels of big (hBK), intermediate (hIK) or small (rSK3) conductance were c... more Ca 2+ -activated K + channels of big (hBK), intermediate (hIK) or small (rSK3) conductance were coexpressed with aquaporin 1 (AQP1) in Xenopus laevis oocytes. hBK channels were activated by depolarization, whereas hIK and rSK3 channels were activated by direct injection of Ca 2+ or Cd 2+ into the oocyte cytoplasm, before the oocytes were subjected to hyperosmolar or hypoosmolar (±50 mOsm mannitol) challenges. In all cases, the oocytes responded rapidly to the osmotic changes with shrinkage or swelling and the effects on the K + currents were measured. hIK and rSK3 currents were highly sensitive to volume changes and increased immediately to 178% (hIK) or 165% (rSK3) of control in response to swelling and decreased to 64% (hIK) or 61% (rSK3) of control after shrinkage. These responses were dependent on the channels being pre-activated and were almost totally abolished after injection of cytochalasin D into the oocyte cytoplasm (final concentration: 1 µM). In contrast, hBK channels showed only a minor sensitivity to volume changes; the hBK channel activity decreased approximately 20% after swelling and increased approximately 20% after shrinkage. The opposite effects of volume changes on hIK/rSK3 and hBK channels suggest that the significant stimulation of hIK and rSK3 channels during swelling is not mediated by changes in intracellular Ca 2+ , but rather through interactions with the cytoskeleton, provided that a sufficient basal concentration of intracellular Ca 2+ or Cd 2+ is present.

Pflügers Archiv - European Journal of Physiology, 2009

It is unclear how ammonia is transported by proteins from the Amt/Mep/Rh superfamily. We investig... more It is unclear how ammonia is transported by proteins from the Amt/Mep/Rh superfamily. We investigated this for the ammonium transporter TaAMT1;1 from wheat expressed in Xenopus oocytes by two-electrode voltage clamp and radio-labeled uptakes. Inward currents were activated by NH (4) (+) or methylammonium ions (MeA(+)). Importantly, currents increased fivefold when the external pH was decreased from 7.4 to 5.5; this type of pH dependence is unique and is a strong indication of NH (4) (+) or MeA(+) transport. This was confirmed by the close correlation between the uptake of radio-labeled MeA(+) and MeA(+)-induced currents. Homology models of members of the Amt/Mep/Rh superfamily exhibited major divergences in their cytoplasmic regions. A point mutation in this region of TaAMT1;1 abolished the pH sensitivity and decreased the apparent affinities for NH (4) (+) and MeA(+). We suggest a model where NH (4) (+) is transported as NH(3) and H(+) via separate pathways but the latter two recombine before leaving the protein.

Neuroscience Letters, 2009

The K+ channels Kir4.1 and Kir4.1-Kir5.1 are expressed in the glial cells of the CNS and are invo... more The K+ channels Kir4.1 and Kir4.1-Kir5.1 are expressed in the glial cells of the CNS and are involved in regulation of the K+ homeostasis. Several studies have shown that Kir4.1 channels are co-localized with aquaporins (AQP4) in the glial endfeet, and a putative functional coupling between the Kir channels and aquaporins is therefore debated. To test a possible volume-sensitivity of the Kir channels, the Kir4.1 or Kir4.1-Kir5.1 channels were expressed in Xenopus oocytes with or without co-expression of aquaporins and subsequently exposed to cell volume alterations. Our results show an increase in Kir4.1 and Kir4.1-Kir5.1 currents upon swelling of the oocytes and a reduction in the current when the oocytes were shrunk. The volume-dependent changes in channel activity were not due to changes in the kinetics of the channels. These findings implicate a putative functional interaction between the Kir channels and aquaporins via small, fast cell volume changes in the glial cells.

Neurochemical Research, 2012

An important feature of neuronal signalling is the increased concentration of K(+) in the extrace... more An important feature of neuronal signalling is the increased concentration of K(+) in the extracellular space. The K(+) concentration is restored to its original basal level primarily by uptake into nearby glial cells. The molecular mechanisms by which K(+) is transferred from the extracellular space into the glial cell are debated. Although spatial buffer currents may occur, their quantitative contribution to K(+) clearance is uncertain. The concept of spatial buffering of K(+) precludes intracellular K(+) accumulation and is therefore (i) difficult to reconcile with the K(+) accumulation repeatedly observed in glial cells during K(+) clearance and (ii) incompatible with K(+)-dependent glial cell swelling. K(+) uptake into non-voltage clamped cultured glial cells is carried out by the Na(+)/K(+)-ATPase and the Na(+)/K(+)/Cl(-) cotransporter in combination. In brain slices and intact optic nerve, however, only the Na(+)/K(+)-ATPase has been demonstrated to be involved in stimulus-evoked K(+) clearance. The glial cell swelling associated with K(+) clearance is prevented under conditions that block the activity of the Na(+)/K(+)/Cl(-) cotransporter. The Na(+)/K(+)/Cl(-) cotransporter is activated by increased K(+) concentration and cotransports water along with its substrates. It thereby serves as a K(+)-dependent molecular water pump under conditions of increased extracellular K(+) load.

Gap junction channels are composed of two hemichannels (connexons) from neighboring cells. Undock... more Gap junction channels are composed of two hemichannels (connexons) from neighboring cells. Undocked connexons have been proposed to form open channels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules with masses lower than 1 kDa. A range of physiologically relevant molecules, smaller than the assumed molecular cutoff of 1 kDa, have therefore been proposed to permeate connexin 43 (Cx43) in its hemichannel configuration. However, the permeability profile of Cx43 hemichannels remains unresolved. Exposure of Cx43-expressing Xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. In spite thereof, a range of smaller biological molecules, such as water, glutamate, lactate, and glucose, did not gain detectable access through the pore whereas ATP did. In contrast, permeability of glutamate, glucose and ATP was...

Gap-junctions are intracellular channels connecting the cytoplasms of a variety of mammalian cell... more Gap-junctions are intracellular channels connecting the cytoplasms of a variety of mammalian cells: Adjacent connexons/hemichannels from two opposing cells dock in the extracellular space to form the communicating gap junction channel. It has become increasingly evident that connexons appear unopposed in the plasma membrane, and that opening of these likely occur under pathophysiological conditions or experimental condition mimicking those. Due to its large-diameter central pore (15 Å; Unger et al. 1999), opening of hemichannels has been suggested leading to release of substances which may be harmful to the cell itself and/or surrounding cells. The study of connexin 43 (Cx43) hemichannels/connexons in native settings is challenged by the growing list of channels (such as P2X-receptors and pannexins) regulated by the same stimuli and cross-specificity of the inhibitors. To perform a more biophysical analysis of the permeability profile of Cx43 hemichannels, Cx43 was functionally expr...

Connexons form the Gap junction, connecting adjacent cells. Each Connexon is composed of six Conn... more Connexons form the Gap junction, connecting adjacent cells. Each Connexon is composed of six Connexin subunits (Cx), with Cx43 being the main connexin expressed in astrocytes. The Gap Junction is established when two adjacent Connexons, dock in the extracellular space, forming a pathway for cell to cell communication. Ions and several biologically active molecules such as ATP/ADP and IP3 pass between cells through Gap Junctions allowing cell to cell communication or synchronization. Additionally, Connexons appear as undocked Hemichannels, potentially forming a channel connecting the cytoplasm with the extracellular space. Hemichannel opening has been reported in vivo following exposure to a multitude of molecular clues, including metabolic inhibition, pH, pathophysiological ion concentrations or loss of membrane polarization; hallmark events of pathofysiological events such as ischemia or spreading depression. It has been speculated that opening of hemichannels allow passage of subs...

Wiley Interdisciplinary Reviews: Membrane Transport and Signaling, 2012

Water transport across cell membranes is central to most physiological functions. About 200 L of ... more Water transport across cell membranes is central to most physiological functions. About 200 L of water move across epithelial cells each day in humans in order to maintain whole-body homeostasis; water transport in and out of organs such as the brain and eye are of major clinical importance. It is well established that the water transport is driven by ion transport, but how? Osmosis is not always the answer: water can be transported against considerable osmotic gradients, apparently without any external osmotic or hydrostatic driving forces. It is generally accepted that cotransporters of the symport type play a key role for the coupling between ion and water fluxes. Models of coupling are either molecular or based on unstirred layer effects, and can be distinguished by their response time: for molecular models, water transport follows changes of substrate transport instantaneously; in unstirred layer models there is a delay while the osmolarity changes in the solutions surrounding the cotransport protein. For cotransporters expressed heterologously in Xenopus oocytes, influx of water can be detected about 1 second after initiation of cotransport of ions and other substrates. This is 20 times faster than expected (and observed) for unstirred layer effects. Water transport in cotransporters is best explained by a molecular model in which ion and water fluxes are coupled by a mechanism within the protein. This would also clarify how cotransporters exploit the free energy in the ion fluxes for the uphill transport of water.

Biochemical Society Transactions, 2015

Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardia... more Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardiac and brain function. Gap junctions are formed by the docking of two hemichannels from neighbouring cells. Undocked Cx43 hemichannels can upon different stimuli open towards the extracellular matrix and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current knowledge of protein kinase C (PKC)-dependent regulation of Cx43 and discuss the divergent results.

The Journal of physiology, 2012

The NKCC1 and NKCC2 isoforms of the mammalian Na⁺–K⁺–2Cl⁻ cotransporter were expressed in Xenopus... more The NKCC1 and NKCC2 isoforms of the mammalian Na⁺–K⁺–2Cl⁻ cotransporter were expressed in Xenopus oocytes and the relation between external ion concentration and water fluxes determined.Water fluxes were determined from changes in the oocytes volume and ion fluxes from 86Rb+ uptake. Isotonic increases in external K⁺ concentration elicited abrupt inward water fluxes in NKCC1; the K⁺ dependence obeyed one-site kinetics with a K₀.₅ of 7.5 mM. The water fluxes were blocked by bumetanide, had steep temperature dependence and could proceed uphill against an osmotic gradient of 20 mosmol l⁻¹. A comparison between ion and water fluxes indicates that 460 water molecules are cotransported for each turnover of the protein. In contrast, NKCC2 did not support water fluxes.Water transport in NKCC1 induced by increases in the external osmolarity had high activation energy and was blocked by bumetanide. The osmotic effects of NaCl were smaller than those of urea and mannitol. This supports the noti...

International review of cytology, 2002

Molecular water pumps are membrane proteins of the cotransport type in which a flux of water is c... more Molecular water pumps are membrane proteins of the cotransport type in which a flux of water is coupled to substrate fluxes by a mechanism within the protein. Free energy can be exchanged between the fluxes. Accordingly, the flux of water may be relatively independent of the external water chemical potential and can even proceed uphill. In short, water is being cotransported. The evidence for water cotransport is reviewed with particular emphasis on electrogenic cotransporters expressed in Xenopus oocytes under voltage clamped conditions. Phenomena such as uphill water transport, tight coupling between water transport and clamp current, cotransport of small hydrophilic molecules, and shifts in reversal potentials with osmolarity are discussed with examples from the Na+/glutamate and Na+/glucose cotransporters. Unstirred layers and electrode artifacts as alternative explanations for such cotransport can be ruled out for both experimental and theoretical reasons. Indeed, substrate flu...

International review of cytology, 2002

Three kinds of membrane proteins have been shown to have water channels properties: the aquaporin... more Three kinds of membrane proteins have been shown to have water channels properties: the aquaporins, the cotransporters, and the uniports. A molecular-kinetic description of water transport in pores is compared to analytical models based on macroscopic parameters such as pore diameter and length. The use and limitations of irreversible thermodynamics is discussed. Experimental data on water and solute permeability in aquaporins are reviewed. No unifying transport model based on macroscopic parameters can be set up; for example, there is no correlation between solute diameter and permeability. Instead, the influence of hydrogen bonds between solute and pore, and the pH dependence of permeability, point toward a model based upon chemical interactions. The atomic model for AQP1 based on electron crystallographic data defines the dimensions and chemical nature of the aqueous pore. These structural data combined with quantum mechanical modeling and computer simulation might result in a re...

Advances in Experimental Medicine and Biology, 2005

... Morten Grunnet, Thomas Jespersen, Nanna K. Jørgensen, Nanna MacAulay, Nicole Schmitt, Olaf Po... more ... Morten Grunnet, Thomas Jespersen, Nanna K. Jørgensen, Nanna MacAulay, Nicole Schmitt, Olaf Pongs, Henrik S. Jensen, Søren-Peter Olesen and Dan A. Klaerke* 1. ABSTRACT 1 ... Kenneth BE Gagnon, Peter K. Lauf, and Robert EW Fyffe* 1. INTRODUCTION ...

Journal of Biological Chemistry, 2014

The permeability and physiological role of several large-pore (hemi)channels are unresolved. Resu... more The permeability and physiological role of several large-pore (hemi)channels are unresolved. Results: Large-pore (hemi)channels, when heterologously expressed, display isoformspecific permeability and gating for ions and fluorescent dyes. Conclusion: Large-pore channels have isoformspecific transport characteristics that can be used for their identification. Significance: Although large-pore channels have characteristic properties in overexpression systems, these properties may be undetectable in native cells.

The Journal of Physiology, 2003

The Journal of Physiology, 2001

Cotransporters are membrane proteins in which the transmembrane fluxes of various substrates are ... more Cotransporters are membrane proteins in which the transmembrane fluxes of various substrates are coupled: the downhill flux of one substrate energizes the uphill flux of another. Evidence is accruing, however, that cotransporters, in addition to their recognized functions, also play a direct role in water transport. All cotransporters of the symport type tested so far exhibit cotransport of water, in which a fixed number of water molecules are translocated together with non-aqueous substrates by a process that is independent of external parameters. In addition, the cotransporters can function as passive water channels. The K + -Cl _ and H + -lactate cotransporters have been found to cotransport 500 water molecules per turnover . The human Na + -glucose cotransporter, the rabbit Na + -glucose cotransporter and the Na + -dicarboxylate cotransporter cotransport 210, 390 and 175 water molecules per turnover, respectively . Osmotic water transport has been described in detail for the Na + -glucose and the Na + -GABA transporters ).

The Journal of Physiology, 2013

We test a novel model of osmosis in aquaporins. • A solute molecule present at the pore mouth can... more We test a novel model of osmosis in aquaporins. • A solute molecule present at the pore mouth can be reflected or permeate the pore; we propose that only reflected molecules induce osmotic water transport, while permeating molecules give rise to no water transport. • We tested a range of channel geometries using aquaporins AQP1 and AQP9 and mutants thereof; the aquaporins were expressed in Xenopus oocytes. Osmotic gradients were generated by solutes of molecular weights in the range 45-595 Daltons. The reflected fraction of a given solute was estimated optically and compared to the permeability obtained from uptakes of radio-labelled solutes. • In accordance with our model there was a linear relationship between solute permeability and reflection coefficient: solutes with high permeability had low reflection coefficients and vice versa. • We found no evidence for significant coupling between water and solute fluxes inside the pore. Abstract We test a novel, stochastic model of osmotic water transport in aquaporins. A solute molecule present at the pore mouth can either be reflected or permeate the pore. We assume that only reflected solute molecules induce osmotic transport of water through the pore, while permeating solute molecules give rise to no water transport. Accordingly, the rate of water transport is proportional to the reflection coefficient σ, while the solute permeability, P S , is proportional to 1 -σ. The model was tested in aquaporins heterologously expressed in Xenopus oocytes. A variety of aquaporin channel sizes and geometries were obtained with the two aquaporins AQP1 and AQP9 and mutant versions of these. Osmotic water transport was generated by adding 20 mM of a range of different-sized osmolytes to the outer solution. The osmotic water permeability and the reflection coefficient were measured optically at high resolution and compared to the solute permeability obtained from short-term uptake of radio-labelled solute under isotonic conditions. For each type of aquaporin there was a linear relationship between solute permeability and reflection coefficient, in accordance with the model. We found no evidence for coupling between water and solute fluxes in the pore. In confirmation of molecular dynamic simulations, we conclude that the magnitude of the osmotic water permeability and the reflection coefficient are determined by processes at the arginine selectivity filter located at the outward-facing end of the pore.

The Journal of Physiology, 2002

The Journal of Physiology, 2002

The Journal of Physiology, 2006

The glucose transporter GLUT2 has been shown to also transport water. In the present paper we inv... more The glucose transporter GLUT2 has been shown to also transport water. In the present paper we investigated the relation between sugar and water transport in human GLUT2 expressed in Xenopus oocytes. Sugar transport was determined from uptakes of non-metabolizable glucose analogues, primarily 3-O-methyl-D-glucopyranoside; key experimental results were confirmed using D(+)-glucose. Water transport was derived from changes in oocyte volume monitored at a high resolution (20 pl, 1 s). Expression of GLUT2 induced a sugar permeability, P S , of about 5 × 10 −6 cm s −1 and a passive water permeability, L p , of 5.5 × 10 −5 cm s −1 . Accordingly, the passive water permeability of a GLUT2 protein is about 10 times higher than its sugar permeability. Both permeabilities were abolished by phloretin. Isosmotic addition of sugar to the bathing solution (replacing mannitol) induced two parallel components of water influx in GLUT2, one by osmosis and one by cotransport. The osmotic driving force arose from sugar accumulation at the intracellular side of the membrane and was given by an intracellular diffusion coefficient for sugar of 10 −6 cm 2 s −1 , one-fifth of the free solution value. The diffusion coefficient was determined in oocytes coexpressing GLUT2 and the water channel AQP1 where water transport was predominantly osmotic. By the cotransport mechanism about 35 water molecules were transported for each sugar molecule by a mechanism within the GLUT2. These water molecules could be transported uphill, against an osmotic gradient, energized by the flux of sugar. This capacity for cotransport is 10 times smaller than that of the Na + -coupled glucose transporters (SGLT1). The physiological role of GLUT2 for intestinal transport under conditions of high luminal sugar concentrations is discussed.

Pflügers Archiv, 2002

Ca 2+ -activated K + channels of big (hBK), intermediate (hIK) or small (rSK3) conductance were c... more Ca 2+ -activated K + channels of big (hBK), intermediate (hIK) or small (rSK3) conductance were coexpressed with aquaporin 1 (AQP1) in Xenopus laevis oocytes. hBK channels were activated by depolarization, whereas hIK and rSK3 channels were activated by direct injection of Ca 2+ or Cd 2+ into the oocyte cytoplasm, before the oocytes were subjected to hyperosmolar or hypoosmolar (±50 mOsm mannitol) challenges. In all cases, the oocytes responded rapidly to the osmotic changes with shrinkage or swelling and the effects on the K + currents were measured. hIK and rSK3 currents were highly sensitive to volume changes and increased immediately to 178% (hIK) or 165% (rSK3) of control in response to swelling and decreased to 64% (hIK) or 61% (rSK3) of control after shrinkage. These responses were dependent on the channels being pre-activated and were almost totally abolished after injection of cytochalasin D into the oocyte cytoplasm (final concentration: 1 µM). In contrast, hBK channels showed only a minor sensitivity to volume changes; the hBK channel activity decreased approximately 20% after swelling and increased approximately 20% after shrinkage. The opposite effects of volume changes on hIK/rSK3 and hBK channels suggest that the significant stimulation of hIK and rSK3 channels during swelling is not mediated by changes in intracellular Ca 2+ , but rather through interactions with the cytoskeleton, provided that a sufficient basal concentration of intracellular Ca 2+ or Cd 2+ is present.

Pflügers Archiv - European Journal of Physiology, 2009

It is unclear how ammonia is transported by proteins from the Amt/Mep/Rh superfamily. We investig... more It is unclear how ammonia is transported by proteins from the Amt/Mep/Rh superfamily. We investigated this for the ammonium transporter TaAMT1;1 from wheat expressed in Xenopus oocytes by two-electrode voltage clamp and radio-labeled uptakes. Inward currents were activated by NH (4) (+) or methylammonium ions (MeA(+)). Importantly, currents increased fivefold when the external pH was decreased from 7.4 to 5.5; this type of pH dependence is unique and is a strong indication of NH (4) (+) or MeA(+) transport. This was confirmed by the close correlation between the uptake of radio-labeled MeA(+) and MeA(+)-induced currents. Homology models of members of the Amt/Mep/Rh superfamily exhibited major divergences in their cytoplasmic regions. A point mutation in this region of TaAMT1;1 abolished the pH sensitivity and decreased the apparent affinities for NH (4) (+) and MeA(+). We suggest a model where NH (4) (+) is transported as NH(3) and H(+) via separate pathways but the latter two recombine before leaving the protein.

Neuroscience Letters, 2009

The K+ channels Kir4.1 and Kir4.1-Kir5.1 are expressed in the glial cells of the CNS and are invo... more The K+ channels Kir4.1 and Kir4.1-Kir5.1 are expressed in the glial cells of the CNS and are involved in regulation of the K+ homeostasis. Several studies have shown that Kir4.1 channels are co-localized with aquaporins (AQP4) in the glial endfeet, and a putative functional coupling between the Kir channels and aquaporins is therefore debated. To test a possible volume-sensitivity of the Kir channels, the Kir4.1 or Kir4.1-Kir5.1 channels were expressed in Xenopus oocytes with or without co-expression of aquaporins and subsequently exposed to cell volume alterations. Our results show an increase in Kir4.1 and Kir4.1-Kir5.1 currents upon swelling of the oocytes and a reduction in the current when the oocytes were shrunk. The volume-dependent changes in channel activity were not due to changes in the kinetics of the channels. These findings implicate a putative functional interaction between the Kir channels and aquaporins via small, fast cell volume changes in the glial cells.