Ian Forster
University of Zurich, Switzerland, Institute of Physiology, Faculty Member
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Abstract A multiple-electrode cochlear implant has been developed at the University of Melbourne to help patients with profound hearing loss and residual auditory nerve fibers comprehend speech. The device has been designed to enable... more
Abstract A multiple-electrode cochlear implant has been developed at the University of Melbourne to help patients with profound hearing loss and residual auditory nerve fibers comprehend speech. The device has been designed to enable electrical stimulation of ...
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A multiple-electrode hearing prosthesis for cochlea implantation in deaf patients has been developed at the University of Melbourne. It has been designed as a multiple-electrode implant to provide the best chance of enabling patients to... more
A multiple-electrode hearing prosthesis for cochlea implantation in deaf patients has been developed at the University of Melbourne. It has been designed as a multiple-electrode implant to provide the best chance of enabling patients to understand speech. It has been shown that an electrode array can be threaded along the coills of the inner ear close to residual auditory nerves. Experimental studies have indicated that the long-term implantation of the array will not lead to significant degeneration of auditory nerve fibres. Loss of platinum from the stimulating electrodes can be minimized with a biphasic constant current pulse, where the first phase is negative with respect to ground. The receiver-stimulator component has also been designed to provide 10-15 channels of stimulation. Furthermore, the phase and amplitude of the stimuli to individual electrodes can be varied to enable the localization of the electrical fields to discrete groups of nerve fibres, and the correct method of frequency and intensity coding to be determined. Finally, the device should be used in the first instance for a specially selected group of adults who are post-lingually deaf.
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ABSTRACT
The rat renal Na(+)/P(i) cotransporter (NaP(i)-IIa) contains 12 native cysteines. When individually replaced by a serine, none appears essential for proper expression and function. Nevertheless, the formation of one essential cysteine... more
The rat renal Na(+)/P(i) cotransporter (NaP(i)-IIa) contains 12 native cysteines. When individually replaced by a serine, none appears essential for proper expression and function. Nevertheless, the formation of one essential cysteine bridge (C5/C6), together with a postulated second bridge, is necessary. To determine the minimum cysteine residues required for functional NaP(i)-IIa, with the goal of generating a Cys-less backbone for structure-function studies, mutants were constructed in which multiple endogenous cysteines were replaced by serines in different combinations. In Xenopus oocytes, most mutants were functional, except those where cysteine pairs C4/C9, C4/C12 or C9/C12 were simultaneously deleted. This suggested that one of these pairs could form the second cysteine bridge essential for expression and/or protein function. Up to eight cysteines could therefore be removed to give a functional Cys-reduced NaP(i)-IIa with activity and kinetics comparable to the wild-type (WT...
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Intrasequence comparison of the type IIa Na(+)-P(i) cotransport protein revealed two regions with high similarity in the first intracellular (ICL-1) and third extracellular (ECL-3) loops. Because the ECL-3 loop contains functionally... more
Intrasequence comparison of the type IIa Na(+)-P(i) cotransport protein revealed two regions with high similarity in the first intracellular (ICL-1) and third extracellular (ECL-3) loops. Because the ECL-3 loop contains functionally important sites that have been identified by cysteine scanning, we applied this method to corresponding sites in the ICL-1 loop. The accessibility of novel cysteines by methanethiosulfonate reagents was assayed electrophysiologically. Mutants N199C and V202C were fully inhibited after methanethiosulfonate ethylammonium exposure, whereas other mutants showed marginal reductions in cotransport function. None showed significant functional loss after exposure to impermeant methanethiosulfonate ethyltrimethylammonium, which suggested a sidedness of Cys modification. Compared with the wild-type (WT), mutant A203C showed altered Na(+) leak kinetics, whereas N199C exhibited decreased apparent substrate affinities. To delineate the role of residue N199 in conferr...
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Characterization of the electrophysiological properties of electrogenic cotransporter function in terms of both steady-state and pre-steady-state kinetics provides essential information for both model generation and identification of... more
Characterization of the electrophysiological properties of electrogenic cotransporter function in terms of both steady-state and pre-steady-state kinetics provides essential information for both model generation and identification of partial reactions in the transport cycle which play a critical role in determining kinetic parameters. Furthermore, the voltage dependence of the apparent affinity constants obtained from the steady state serves to emphasize the
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The rate of proximal tubular reabsorption of phosphate (P(i)) is a major determinant of P(i) homeostasis. Deviations of the extracellular concentration of P(i) are corrected by many factors that control the activity of Na-P(i) cotransport... more
The rate of proximal tubular reabsorption of phosphate (P(i)) is a major determinant of P(i) homeostasis. Deviations of the extracellular concentration of P(i) are corrected by many factors that control the activity of Na-P(i) cotransport across the apical membrane. In this review, we describe the regulation of proximal tubule P(i) reabsorption via one particular Na-P(i) cotransporter (the type IIa cotransporter) by parathyroid hormone (PTH) and dietary phosphate intake. Available data indicate that both factors determine the net amount of type IIa protein residing in the apical membrane. The resulting change in transport capacity is a function of both the rate of cotransporter insertion and internalization. The latter process is most likely regulated by PTH and dietary P(i) and is considered irreversible since internalized type IIa Na-P(i) cotransporters are subsequently routed to the lysosomes for degradation.
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A sodium-dependent phosphate transporter (type II Na/Pi-cotransporter) was isolated which is expressed in apical membranes of proximal tubules and exhibits transport characteristics similar as described for renal reabsorption of... more
A sodium-dependent phosphate transporter (type II Na/Pi-cotransporter) was isolated which is expressed in apical membranes of proximal tubules and exhibits transport characteristics similar as described for renal reabsorption of phosphate. Type II associated Na/Pi-cotransport is electrogenic and results obtained by electrophysiological measurements support a transport model having a stoichiometry of 3 Na+/HPO4=. Changes of transport such as by parathyroid hormone and altered dietary intake of phosphate correlate with changes of the number of type II cotransporters in the apical membrane. These data suggest that the type II Na/Pi-cotransporter represents the main target for physiological and pathophysiological regulation.
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A type II Na/Pi-cotransporter located in the brush border membrane is the rate limiting and physiologically regulated step in proximal tubular phosphate (Pi) reabsorption. In states of altered Pi-reabsorption [for example, in response to... more
A type II Na/Pi-cotransporter located in the brush border membrane is the rate limiting and physiologically regulated step in proximal tubular phosphate (Pi) reabsorption. In states of altered Pi-reabsorption [for example, in response to parathyroid hormone (PTH) and to altered dietary intake of Pi or as a consequence of genetic abnormalities], brush border expression of the type II Na/Pi-cotransporter is accordingly modified. PTH initiates a regulatory cascade leading to membrane retrieval, followed by lysosomal degradation of this transporter; recovery from inhibition requires its de novo synthesis. Pi-deprivation leads to an increased brush border expression of transporters that does not appear to require de novo synthesis in the short term. Pi-overload leads to membrane retrieval and degradation of transporters. Finally, in animals with genetically altered Pi-handling (Hyp; Gy) the brush border membrane expression of the type II Na/Pi-cotransporter is also reduced, suggesting th...
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Phosphate (Pi) homeostasis is achieved by adjusting the intestinal absorption and the renal excretion. Renal proximal reabsorption of Pi is regulated by controlling the amount of NaPi-IIa cotransporters in the brush border membrane of... more
Phosphate (Pi) homeostasis is achieved by adjusting the intestinal absorption and the renal excretion. Renal proximal reabsorption of Pi is regulated by controlling the amount of NaPi-IIa cotransporters in the brush border membrane of proximal tubules. Therefore, the understanding of the molecular mechanisms that control the apical expression of NaPi-IIa is required to have a full picture of how phosphate balancing takes place. In this review we will summarize our present knowledge about the mechanisms involved in the regulation of the apical expression and membrane retrieval of this family of transporters.
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A key process in overall P(i)-homeostasis is renal proximal tubular reabsorption of inorganic phosphate (P(i)), which involves secondary active sodium/phosphate (Na(+)/P(i)) cotransport reabsorption at the brush border membrane. Among the... more
A key process in overall P(i)-homeostasis is renal proximal tubular reabsorption of inorganic phosphate (P(i)), which involves secondary active sodium/phosphate (Na(+)/P(i)) cotransport reabsorption at the brush border membrane. Among the two different molecularly identified Na(+)/P(i) cotransporters, the type-IIa Na(+)/P(i) cotransporter (NaPi-IIa) accounts for up to 70% of brush border membrane transport. Regulation of renal P(i) reabsorption centers around brush border membrane insertion and retrieval of transporter protein under the influence of hormonal and nonhormonal factors. Immunohistochemical and fluorescence techniques have provided new insights into the tissue distribution and the regulation processes. The intrinsic electrogenicity of NaPi-IIa, has allowed detailed studies of the transport kinetics of NaPi-IIa and, combined with mutagenesis methods, structure-function information at the protein level is emerging.
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Research Interests: Kinetics, Protein Structure and Function, Transport Properties, Kidney, Case Study, and 15 moreAnimals, Real Time, Protein Secondary Structure Prediction, Sodium, Substrate Specificity, Phosphates, Protein Conformation, Amino Acid Sequence, Ion Transport, Membrane Protein, Heterologous Expression, Structure activity Relationship, Structure Function, Hydrogen-Ion Concentration, and Biochemistry and cell biology
The effect of tetrodotoxin (TTX) on the sodium gating current in the squid giant axon was examined by recording the current that flowed at the pulse potential at which the ionic current fell to zero, first in the absence and then in the... more
The effect of tetrodotoxin (TTX) on the sodium gating current in the squid giant axon was examined by recording the current that flowed at the pulse potential at which the ionic current fell to zero, first in the absence and then in the presence of TTX. The addition of 1 microM TTX to the bathing solution had no consistent effect on the size of the initial peak of the gating current, but resulted in small changes in the timecourse of its subsequent relaxation which were mainly caused by a reduction of about one quarter in the component that has a delayed onset and may possibly arise from changes in the state of ionization of groups in the channel wall when the lumen fills with water. Our findings suggest that the binding of TTX at the outer face of the sodium channel does not interfere with the mechanisms of activation and inactivation by the voltage sensors, but has an allosteric effect on the access of internal cations to the inside of the channel.
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A critical study has been made of the characteristics of the kinetic components of the sodium gating current in the squid giant axon, of which not less than five can be resolved. In addition to the principal fast component Ig2, there are... more
A critical study has been made of the characteristics of the kinetic components of the sodium gating current in the squid giant axon, of which not less than five can be resolved. In addition to the principal fast component Ig2, there are two components of appreciable size that relax at an intermediate rate, Ig3 alpha and Ig 3 beta. Ig3 alpha has a fast rise, and is present over the whole range of negative test potentials. Ig3 beta is absent below -40 mV, exhibits a delayed onset and disappears on inactivation of the sodium system. There are also two smaller components, Ig1 and Ig4, with very fast and much slower relaxation time constants, respectively.
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Comparisons were made between families of ion currents recorded in voltage-clamped squid axons dialysed with 20 mM NaF and 330 mM CsF or TMAF, and bathed in a solution in which four fifths of the Na was replaced by Tris. The permeability... more
Comparisons were made between families of ion currents recorded in voltage-clamped squid axons dialysed with 20 mM NaF and 330 mM CsF or TMAF, and bathed in a solution in which four fifths of the Na was replaced by Tris. The permeability coefficient PNa,fast for the fast-inactivating current in the initial open state was calculated as a function of test potential from the size of the initial peak of INa. The permeability coefficient PNa,non for the non-inactivating open state was calculated from the steady-state INa that persisted until the end of the test pulse. Dialysis with TMA had no direct effect on the QV curve for gating charge. The reversal potential for INa,non was always lower than that for INa,fast, the mean difference being about -9 mV when dialysing with Cs, but only about -1 mV with TMA. Except close to threshold, PNa,fast was roughly halved by dialysis with TMA as compared with Cs, but PNa,non was substantially increased. The time constant tau h inactivation of the sodium system was slightly increased during dialysis with TMA in place of Cs, and there were small shifts in the steady-state inactivation curve, but the rate of recovery from inactivation was not measurably altered. The flattening off of the tau h curve at increasingly positive test potentials corresponded to a steady reduction of the apparent inactivation charge until a value of about 0.2e was reached for pulses to 100 mV. The instantaneous I-V relationship in the steady state was also investigated. The results have a useful bearing on the effects of dialysis with TMA, on the differences between the initial and steady open states of the sodium channel, and on the relative voltage-dependences of the transitions in each direction between the resting and inactivated states.
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Homeostasis of inorganic phosphate (P(i)) is primarily an affair of the kidneys. Reabsorption of the bulk of filtered P(i) occurs along the renal proximal tubule and is initiated by apically localized Na(+)-dependent P(i) cotransporters.... more
Homeostasis of inorganic phosphate (P(i)) is primarily an affair of the kidneys. Reabsorption of the bulk of filtered P(i) occurs along the renal proximal tubule and is initiated by apically localized Na(+)-dependent P(i) cotransporters. Tubular P(i) reabsorption and therefore renal excretion of P(i) is controlled by a number of hormones, including phosphatonins, and metabolic factors. In most cases, regulation of P(i) reabsorption is achieved by changing the apical abundance of Na(+)/Pi cotransporters. The regulatory mechanisms involve various signaling pathways and a number of proteins that interact with Na(+)/P(i) cotransporters.
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Research Interests: Physiology, Thermodynamics, Electrophysiology, Kinetics, Lithium, and 18 morePatch-clamp and imaging techniques, Mice, Female, Animals, Medical Physiology, Steady state, European, Amino Acids, Phenylalanine, Sodium, Xenopus laevis, Amino Acid Profile, Transfection, Glycine, Oocytes, Voltage Clamp, Hydrogen-Ion Concentration, and Plasma Membrane
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The SLC34 family of sodium-driven phosphate cotransporters comprises three members: NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). These transporters mediate the translocation of divalent inorganic phosphate (HPO4 (2-))... more
The SLC34 family of sodium-driven phosphate cotransporters comprises three members: NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). These transporters mediate the translocation of divalent inorganic phosphate (HPO4 (2-)) together with two (NaPi-IIc) or three sodium ions (NaPi-IIa and NaPi-IIb), respectively. Consequently, phosphate transport by NaPi-IIa and NaPi-IIb is electrogenic. NaPi-IIa and NaPi-IIc are predominantly expressed in the brush border membrane of the proximal tubule, whereas NaPi-IIb is found in many more organs including the small intestine, lung, liver, and testis. The abundance and activity of these transporters are mostly regulated by changes in their expression at the cell surface and are determined by interactions with proteins involved in scaffolding, trafficking, or intracellular signaling. All three transporters are highly regulated by factors including dietary phosphate status, hormones like parathyroid hormone, 1,25-OH2 vitamin D3 or FGF23, electrolyte, and acid-base status. The physiological relevance of the three members of the SLC34 family is underlined by rare Mendelian disorders causing phosphaturia, hypophosphatemia, or ectopic organ calcifications.
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The ubiquitous CBS domains, which are found as part of cytoplasmic domains in the ClC family of chloride channels and transporters, have previously been identified as building blocks for regulatory nucleotide-binding sites. Here we report... more
The ubiquitous CBS domains, which are found as part of cytoplasmic domains in the ClC family of chloride channels and transporters, have previously been identified as building blocks for regulatory nucleotide-binding sites. Here we report the structures of the cytoplasmic domain of the human transporter ClC-5 in complex with ATP and ADP. The nucleotides bind to a specific site in the protein. As determined by equilibrium dialysis, the affinities for ATP, ADP and AMP are in the high micromolar range. Point mutations that interfere with nucleotide binding change the transport behavior of a ClC-5 mutant expressed in Xenopus laevis oocytes. Our results establish the structural and energetic basis for the interaction of ClC-5 with nucleotides and provide a framework for future investigations.
Research Interests: Macromolecular X-Ray Crystallography, Energy Metabolism, Patch-clamp and imaging techniques, Biological Sciences, Humans, and 13 moreMutagenesis, Animals, CHEMICAL SCIENCES, Xenopus laevis, Chloride Channel, Amino Acid Sequence, Oocytes, Protein Binding, Ligands, Nucleotides, Adenosine Triphosphate, Adenosine monophosphate, and Adenosine Diphosphate
Renal and small intestinal (re-)absorption contribute to overall phosphate(Pi)-homeostasis. In both epithelia, apical sodium (Na+)/Pi-cotransport across the luminal (brush border) membrane is rate limiting and the target for... more
Renal and small intestinal (re-)absorption contribute to overall phosphate(Pi)-homeostasis. In both epithelia, apical sodium (Na+)/Pi-cotransport across the luminal (brush border) membrane is rate limiting and the target for physiological/pathophysiological alterations. Three different Na/Pi-cotransporters have been identified: (i) type I cotransporter(s)--present in the proximal tubule--also show anion channel function and may play a role in secretion of organic anions; in the brain, it may serve vesicular glutamate uptake functions; (ii) type II cotransporter(s) seem to serve rather specific epithelial functions; in the renal proximal tubule (type Ila) and in the small intestine (type IIb), isoform determines Na+-dependent transcellular Pi-movements; (iii) type III cotransporters are expressed in many different cells/tissues where they could serve housekeeping functions. In the small intestine, alterations in Pi-absorption and, thus, apical expression of IIb protein are mostly in response to longer term (days) situations (altered Pi-intake, levels of 1.25 (OH2) vitamin D3, growth, etc), whereas in renal proximal tubule, in addition, hormonal effects (e.g. Parathyroid Hormone, PTH) acutely control (minutes/hours) the expression of the IIa cotransporter. The type II Na/Pi-cotransporters operate (as functional monomers) in a 3 Na+:1 Pi stoichiometry, including transfer of negatively charged (-1) empty carriers and electroneutral transfers of partially loaded carriers (1 Na+, slippage) and of the fully loaded carriers (3 Na+, 1 Pi). By a chimera (IIa/IIb) approach, and by site-directed mutagenesis (including cysteine-scanning), specific sequences have been identified contributing to either apical expression, PTH-induced membrane retrieval, Na+-interaction or specific pH-dependence of the IIa and IIIb cotransporters. For the COOH-terminal tail of the IIa Na/Pi-cotransporter, several interacting PDZ-domain proteins have been identified which may contribute to either its apical expression (NaPi-Cap1) or to its subapical/lysosomal traffic (NaPi-Cap2).
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The design of an analog interface to a digital audio signal processor (DASP)-video cassette recorder (VCR) system is described. The complete system represents a low-cost alternative to both FM instrumentation tape recorders and... more
The design of an analog interface to a digital audio signal processor (DASP)-video cassette recorder (VCR) system is described. The complete system represents a low-cost alternative to both FM instrumentation tape recorders and multi-channel chart recorders. The interface or DASP input-output unit described in this paper enables the recording and playback of up to 12 analog channels with a maximum of 12 bit resolution and a bandwidth of 2 kHz per channel. Internal control and timing in the recording component of the interface is performed using ROMs which can be reprogrammed to suit different analog-to-digital converter hardware. Improvement in the bandwidth specifications is possible by connecting channels in parallel. A parallel 16 bit data output port is provided for direct transfer of the digitized data to a computer.
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The design of a voltage-clamp system dedicated to recording the fluctuation of sodium currents under non-stationary conditions from a leaflet of cut-open squid axon is presented. The membrane leaflet is mechanically sandwiched between the... more
The design of a voltage-clamp system dedicated to recording the fluctuation of sodium currents under non-stationary conditions from a leaflet of cut-open squid axon is presented. The membrane leaflet is mechanically sandwiched between the apices of two finely machined plexiglass cones which enable fluid access to each side of the membrane and a known area of membrane to be voltage-clamped. The design requirements necessary to achieve satisfactory signal resolution have been assessed in terms of the overall digitising resolution of the ADC hardware and the intrinsic and extrinsic components of the clamp-system noise. Good agreement between the predicted and measured noise performance was found. The clamp system has enabled simultaneous estimates of the single-channel conductance and channel density to be made over a much wider range of experimental conditions than previously possible.
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We have combined a functional assay, surface labeling and immunocytochemical methods to compare total and surface-exposed renal type IIa Na+/Pi cotransporter protein. The wild-type type cotransporter (NaPi-IIa) and its functionally... more
We have combined a functional assay, surface labeling and immunocytochemical methods to compare total and surface-exposed renal type IIa Na+/Pi cotransporter protein. The wild-type type cotransporter (NaPi-IIa) and its functionally comparable cysteine mutant S460C were expressed in Xenopus oocytes. S460C contains a novel cysteine residue that, when modified by preincubation with methanethiosulfonate reagents, leads to complete suppression of cotransport function. This allowed surface labeling of the S460C using MTSEA-Biotin and confirmation by electrophysiology on the same cell. Protein was analyzed by Western blotting before and after streptavidin precipitation and by immunocytochemistry and immunogold electronmicroscopy. MTSEA-Biotin treatment resulted in a complete inhibition of S460C-mediated Na+/Pi-cotransport activity, which indicated that all transporters at the surface were biotinylated. After biotinylation, only a small fraction of total S460C protein was precipitated by streptavidin compared with the total amount of S460C protein detected in the lysate. Light- and electron-microscopy analysis of oocytes showed a large amount of WT and S460C transporter protein beneath the oocyte membrane. These data indicate that the apparent weak labeling efficiencies of surface-biotinylation-based assays of membrane proteins heterologously expressed in oocytes can be related to diminished incorporation of the protein in the oolemma.
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Research Interests: Microbiology, Electrophysiology, Kinetics, Medical Microbiology, Membrane Biology, and 16 morePatch-clamp and imaging techniques, Female, Animals, Temperature, Activation Energy, Steady state, Membrane, Temperature Dependence, Constant Time Delay, Flounder, Xenopus laevis, Oocytes, Voltage Clamp, Charge Distribution, Conformational Change, and Biochemistry and cell biology
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Research Interests: Microbiology, Electrophysiology, Kinetics, Medical Microbiology, Membrane Biology, and 13 moreGene expression, Animals, Steady state, Flounder, Sodium, Xenopus laevis, Phosphates, Oocytes, Voltage Clamp, Proximal Tubule, Hydrogen-Ion Concentration, Electric Conductivity, and Biochemistry and cell biology
The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously... more
The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY402FAM matching the consensus sequence GYXXZ, and Y509RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated 32Pi-uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lack of glycosylation and reduced surface expression of the cotransporter, that are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasma membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that the Y402 is important for the surface expression whereas Y509 for the function of the type II Na/Pi-cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter.
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The effects of the arginine-modifying reagent phenylglyoxal on the kinetics of the type IIa Na + /Pi cotransporter expressed in Xenopus, oocytes were studied by means of 32Pi uptake and electrophysiology. Phenylglyoxal incubation induced... more
The effects of the arginine-modifying reagent phenylglyoxal on the kinetics of the type IIa Na + /Pi cotransporter expressed in Xenopus, oocytes were studied by means of 32Pi uptake and electrophysiology. Phenylglyoxal incubation induced up to 60% loss of cotransport function but only marginally altered the Na+-leak. Substrate activation and pH dependency remained essentially unaltered, whereas the voltage dependency of Pi-induced change in electrogenic response was significantly reduced. Presteady-state charge movements were suppressed and the equilibrium charge distribution was shifted slightly towards hyperpolarizing potentials. Charge movements in the absence of external Na+ were also suppressed, which indicated that the empty-carrier kinetics were modified. These effects were incorporated into an ordered alternating access model for NaPi-IIa, whereby the arginine modification by phenylglyoxal was modeled as altered apparent electrical distances moved by mobile charges, together with a slower rate of translocation of the electroneutral, fully loaded carrier.
Research Interests: Microbiology, Kinetics, Medical Microbiology, Membrane Biology, Enzyme Inhibitors, and 12 morePatch-clamp and imaging techniques, Phosphorus, Computer Simulation, Animals, Xenopus laevis, Reproducibility of Results, Arginine, Sensitivity and Specificity, Oocytes, Charge Distribution, Biochemistry and cell biology, and Gene Expression Regulation
The type IIa Na(+)/P(i), cotransporter (NaPi-IIa) mediates electrogenic transport of three Na(+) and one divalent P(i) ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb... more
The type IIa Na(+)/P(i), cotransporter (NaPi-IIa) mediates electrogenic transport of three Na(+) and one divalent P(i) ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and P(i) transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of P(i) transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the P(i)-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na(+)-dependent P(i) transport. Since electrogenic NaPi-II transports 3 Na(+)/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na(+) binding sites in NaPi-IIa.
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Analysis of rat and mouse proximal tubular brush-border membrane expression of the type IIa Na/P(i)-cotransporter provides evidence for its cleavage in the large extracellular loop (ECL-2). To study functional properties and membrane... more
Analysis of rat and mouse proximal tubular brush-border membrane expression of the type IIa Na/P(i)-cotransporter provides evidence for its cleavage in the large extracellular loop (ECL-2). To study functional properties and membrane distribution of this split NaP(i)-IIa transporter we followed two strategies. In one strategy we expressed the transporter as two complementary parts (p40 and p45) in Xenopus laevis oocytes and as another strategy we cleaved the WT protein with trypsin. Both strategies resulted in a split NaP(i)-IIa protein located in the plasma membrane. The two domains were tied together by a disulfide bridge, most likely involving the cysteines 306 and 334. Surface expression of the NaP(i)-IIa fragments was dependent on the presence of both domains. If both domains were coexpressed, the transporter was functional and transport characteristics were identical to those of the WT-NaP(i)-IIa protein. Corresponding to this, the transporter cleaved by trypsin also retains its transport capacity. These data indicate that cleavage of the type IIa Na/P(i)-cotransporter at ECL-2 is compatible with its cotransport function.
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Research Interests: Microbiology, Electrophysiology, Kinetics, Medical Microbiology, Membrane Biology, and 17 morePatch-clamp and imaging techniques, Xenopus, Humans, Mice, Female, Animals, Steady state, Membrane, Rats, Species Specificity, Oocytes, Voltage Clamp, Recombinant Proteins, Hydrogen-Ion Concentration, Charge Distribution, Small Intestine, and Biochemistry and cell biology
A multiple-electrode cochlear implant comprised of three intracochlear and four extracochlear electrodes is described. A percutaneous plug allowed for direct electrical measurements and presentation of stimuli to selected electrode... more
A multiple-electrode cochlear implant comprised of three intracochlear and four extracochlear electrodes is described. A percutaneous plug allowed for direct electrical measurements and presentation of stimuli to selected electrode combinations. Electrical impedance measurements revealed functions that decreased as frequency increase to about 20 kHz. Absolute impedance magnitude varied across electrodes. Equal loudness contours were similar in shape for all three cochlear electrodes, although dynamic range was significantly reduced for the most apical electrode. Periodicity and place pitch were demonstrated; effects were most salient for low frequency stimuli. A 3-channel sound processor was built and multiple-channel stimulation was compared to single-channel stimulation. Significant performance differences were not found between single-channel and multiple-channel systems for environmental or speech sounds. However, the patient subjectively reported superior quality with the multiple-channel system.
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INTRA-COCHLEAR single-channel electrical stimulation has recently been attempted by Michelson (1971) and by House and Urban (1973). Douek et al.(1977) have described experiments with a single-channel promontory electrode system. It is... more
INTRA-COCHLEAR single-channel electrical stimulation has recently been attempted by Michelson (1971) and by House and Urban (1973). Douek et al.(1977) have described experiments with a single-channel promontory electrode system. It is generally accepted ...
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Research Interests: Physiology, Electrophysiology, Lithium, Patch-clamp and imaging techniques, Female, and 14 moreAnimals, Medical Physiology, Flounder, Sodium, Substrate Specificity, Xenopus laevis, Phosphates, Cysteine, Protein Conformation, Oocytes, Voltage Clamp, Protein Binding, Fluorometry, and General Physiology
Research Interests: Physiology, Electrophysiology, Kinetics, Mutagenesis, Animals, and 16 moreMedical Physiology, Steady state, Rats, Sodium, Xenopus laevis, Phosphates, Cysteine, Alkylation, Amino Acid Sequence, Amino Acid Substitution Rates, Oocytes, Structure activity Relationship, Conformational Change, Chemical Modification, General Physiology, and Protein Transport
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Type II Na/P(i) cotransporters play key roles in epithelial P(i) transport and thereby contribute to overall P(i) homeostasis. Renal proximal tubular brush border membrane expresses the IIa isoform, whereas the IIb isoform is... more
Type II Na/P(i) cotransporters play key roles in epithelial P(i) transport and thereby contribute to overall P(i) homeostasis. Renal proximal tubular brush border membrane expresses the IIa isoform, whereas the IIb isoform is preferentially expressed in small intestinal brush border membrane of mammals. IIa and IIb proteins are predicted to contain eight transmembrane domains with the N- and C-terminal tails facing the cytoplasm. They differ in their pH dependences: the activity of IIa increases at higher pH, whereas the IIb shows no or a slightly opposite pH dependence. To determine the structural domains responsible for the difference in pH sensitivity, mouse IIa and IIb chimeras were constructed, and their pH dependence was characterized. A region between the fourth and fifth transmembrane domains was required for conferring pH sensitivity to the IIa-mediated Na/P(i) cotransport. Sequence comparison (IIa versus IIb) of the third extracellular loops revealed a stretch of three charged amino acids in IIa (REK) replaced by uncharged residues in IIb (GNT). Introduction of the uncharged GNT sequence (by REK) in IIa abolished its pH dependence, whereas introduction of the charged REK stretch in IIb (by GNT) led to a pH dependence similar to IIa. These findings suggest that charged residues within the third extracellular loop are involved in the pH sensitivity of IIa Na/P(i) cotransporter.
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The composition of the functional unit of the rat renal type IIa Na(+)/P(i) cotransporter (NaPi-IIa) was investigated by using two approaches based on the differential sensitivities of the wild type (WT) and mutant S460C proteins to... more
The composition of the functional unit of the rat renal type IIa Na(+)/P(i) cotransporter (NaPi-IIa) was investigated by using two approaches based on the differential sensitivities of the wild type (WT) and mutant S460C proteins to 2-aminoethylmethanethiosulfonate hydrobromide (MTSEA), a charged cysteine modifier. Transport activity of S460C is completely blocked after incubation in MTSEA, whereas that of the WT remains unaffected. First, Xenopus laevis oocytes were coinjected with cRNAs coding for the WT and S460C in different proportions, and the transport inhibition after MTSEA incubation was assayed by electrophysiology. The relationship between MTSEA inhibition and proportion of cRNA was consistent with that for a functional monomer. Second, concatameric proteins were constructed that either comprised two WT proteins (WT-WT), two S460C mutants (S460C-S460C), or one of each (WT-S460C). Western blots of oocytes injected with fusion protein cRNA showed bands at approximately 200 kDa, whereas a main band at approximately 90 kDa was obtained for the WT cRNA alone. The kinetic properties of concatamers were the same as for the single proteins. Transport activity of the WT-WT concatamer was unaffected by MTSEA incubation, fully inhibited for S460C-S460C, but 50% inhibited for WT-S460C. This behavior was also consistent with NaPi-IIa being a functional monomer.
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Using a very low noise voltage clamp technique it has been possible to record from the squid giant axon a slow component of gating current (Ig) during the inactivation phase of the macroscopic sodium current (INa) which was hitherto... more
Using a very low noise voltage clamp technique it has been possible to record from the squid giant axon a slow component of gating current (Ig) during the inactivation phase of the macroscopic sodium current (INa) which was hitherto buried in the baseline noise. In order to examine whether this slow Ig contains gating charge that originates from transitions between the open (O) and the inactivated (I) states, which would indicate a true voltage dependence of inactivation, or whether other transitions contribute charge to slow Ig, a new model independent analysis termed isochronic plot analysis has been developed. From a direct correlation of Ig and the time derivative of the sodium conductance dgNa/dt the condition when only O-I transitions occur is detected. Then the ratio of the two signals is constant and a straight line appears in an isochronic plot of Ig vs. dgNa/dt. Its slope does not depend on voltage or time and corresponds to the quantal gating charge of the O-I transition (qh) divided by the single channel ionic conductance (gamma). This condition was found at voltages above -10 mV up to +40 mV and a figure of 1.21 e- was obtained for qh at temperatures of 5 and 15 degrees C. At lower voltages additional charge from other transitions, e.g. closed to open, is displaced during macroscopic inactivation. This means that conventional Eyring rate analysis of the inactivation time constant tau h is only valid above -10 mV and here the figure for qh was confirmed also from this analysis. It is further shown that most of the present controversies surrounding the voltage dependence of inactivation can be clarified. The validity of the isochronic plot analysis has been confirmed using simulated gating and ionic currents.
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Research Interests: Kinetics, Biophysical Chemistry, Patch-clamp and imaging techniques, Biological Sciences, Animals, and 15 morePhysical sciences, Steady state, CHEMICAL SCIENCES, Flounder, Rats, Sodium, Xenopus laevis, Phosphates, Transfection, Protein isoforms, Ion Transport, Oocytes, Proximal Tubule, Hydrogen-Ion Concentration, and Acids
We have characterized the kinetics of substrate transport in the renal type IIa human sodium-phosphate cotransporter (NaPi-IIa). The transporter was expressed in Xenopus laevis oocytes, and steady-state and pre-steady-state currents and... more
We have characterized the kinetics of substrate transport in the renal type IIa human sodium-phosphate cotransporter (NaPi-IIa). The transporter was expressed in Xenopus laevis oocytes, and steady-state and pre-steady-state currents and substrate uptakes were characterized by voltage-clamp and isotope flux. First, by measuring simultaneous uptake of a substrate (32Pi, 22Na) and charge in voltage-clamped oocytes, we established that the human NaPi-IIa isoform operates with a Na:Pi:charge stoichiometry of 3:1:1 and that the preferred transported Pi species is HPO4(2-). We then probed the complex interrelationship of substrates, pH, and voltage in the NaPi-IIa transport cycle by analyzing both steady-state and pre-steady-state currents. Steady-state current measurements show that the apparent HPO4(2-) affinity is voltage dependent and that this voltage dependency is abrogated by lowering the pH or the Na+ concentration. In contrast, the voltage dependency of the apparent Na+ affinity increased when pH was lowered. Pre-steady-state current analysis shows that Na+ ions bind first and influence the preferred orientation of the transporter in the absence of Pi. Pre-steady-state charge movement was partially suppressed by complete removal of Na+ from the bath, by reducing extracellular pH (both in the presence and absence of Na+), or by adding Pi (in the presence of 100 mM Na). None of these conditions suppressed charge movement completely. The results allowed us to modify previous models for the transport cycle of NaPi-II transporters by including voltage dependency of HPO4(2-) binding and proton modulation of the first Na+ binding step.
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Inorganic phosphate (Pi) is essential for all living organisms. Bound to organic molecules, Pi fulfills structural, metabolic, and signaling tasks. Therefore, cell growth and maintenance depends on efficient transport of Pi across... more
Inorganic phosphate (Pi) is essential for all living organisms. Bound to organic molecules, Pi fulfills structural, metabolic, and signaling tasks. Therefore, cell growth and maintenance depends on efficient transport of Pi across cellular membranes into the intracellular space. Uptake of Pi requires energy because the substrate is transported against its electrochemical gradient. Till recently, 2 major families of physiologically relevant Pi-specific transporters have been identified: the solute carrier families Slc34 and Slc20. Interestingly, phylogenetic links can be detected between prokaryotic and eukaryotic transporters in both families. Because less complex model organisms are often instrumental in establishing paradigms for protein function in human beings, a brief assessment of Slc34 and Slc20 phylogeny is of interest.