Biomedical subjects

Katja Köhler

Publications and source records attributed to Katja Köhler.

7 recordsLinked to original sources

PubMed2005

Tight junction: a co-ordinator of cell signalling and membrane trafficking.

Increasing evidence indicates that the tight junction plays a role in membrane transport. Various signalling and trafficking molecules localize to the sites of cell-cell junctions in epithelial cells, including Rab proteins, a family of small GTPases that regulate different steps of vesicular transport along the endocytic and exocytic pathways. We have recently shown that Rab13 controls protein kinase A activity, demonstrating a clear biochemical and functional link between Rab13 and protein kinase A signalling during tight junction assembly in epithelial cells. The present article focuses on how protein kinase A signalling and protein trafficking events could be integrated at tight junctions in epithelial cells.

Biological Transport↗

PubMed2004

Rab13 regulates PKA signaling during tight junction assembly.

The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell-cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.

Animals↗

PubMed2003

Essential cysteine residues of the type IIa Na+/Pi cotransporter.

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). This construct, like all intermediate mutants and the WT, was insensitive to cysteine-modifying methanethiosulfonate (MTS) reagents. Moreover, by introducing a novel cysteine into the Cys-reduced NaP(i)-IIa at a site functionally important in the WT (Ser-460), the loss of transport function reported for mutant S460C, after exposure to MTS reagents, was recapitulated. This confirmed that the MTS reagent site of action was Cys-460 and that modification of native cysteines does not contribute to S460C behavior.

Animals↗

PubMed2002

Forging the link between structure and function of electrogenic cotransporters: the renal type IIa Na+/Pi cotransporter as a case study.

Electrogenic cotransporters are membrane proteins that use the electrochemical gradient across the cell membrane of a cosubstrate ion, for example Na(+) or H(+), to mediate uphill cotransport of a substrate specific to the transport protein. The cotransport process involves recognition of both cosubstrate and substrate and translocation of each species according to a defined stoichiometry. Electrogenicity implies net movement of charges across the membrane in response to the transmembrane voltage and therefore, in addition to isotope flux assays, the cotransport kinetics can be studied in real-time using electrophysiological methods. As well as the cotransport mode, many cotransporters also display a uniport or slippage mode, whereby the cosubstrate ions translocate in the absence of substrate. The current challenge is to define structure-function relationships by identifying functionally important elements in the protein that confer the transport properties and thus contribute to the ultimate goal of having a 3-D model of the protein that conveys both structural and functional information. In this review we focus on a functional approach to meet this challenge, based on a combination of real-time electrophysiological assays, together with molecular biological and biochemical methods. This is illustrated, by way of example, using data obtained by heterologous expression of the renal Na(+)-coupled inorganic phosphate cotransporter (NaP(i)-IIa) for which structure-function relationships are beginning to emerge.

Amino Acid Sequence↗

PubMed2002

Transport function of the renal type IIa Na+/P(i) cotransporter is codetermined by residues in two opposing linker regions.

Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/P(i) cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region. The effect of cysteine modification by different MTS reagents on two electrogenic transport modes (leak and cotransport) was investigated. MTSEA (2-aminoethyl MTS hydrobromide) and MTSES (MTS ethylsulfonate) led to full inhibition of cotransport and increased the leak, whereas incubation in MTSET (2-[trimethylammonium]ethyl MTS bromide) inhibited only cotransport. The behavior of other double mutants with a cysteine retained at one site and hydrophobic or hydrophilic residues substituted at the other site, indicated that most likely only Cys-460 was modifiable, but the residue at Ala-203 was critical for conferring the leak and cotransport mode behavior. Substrate interaction with the double mutant was unaffected by MTS exposure as the apparent P(i) and Na+ affinities for P(i)-induced currents and respective activation functions were unchanged after cysteine modification. This suggested that the modified site did not interfere with substrate recognition/binding, but prevents translocation of the fully loaded carrier. The time-dependency of cotransport loss and leak growth during modification of the double cysteine mutant was reciprocal, which suggested that the modified site is a kinetic codeterminant of both transport modes. The behavior is consistent with a kinetic model for NaPi-IIa that predicts mutual exclusiveness of both transport modes. Together, these findings suggest that parts of the opposing linker regions are associated with the NaPi-IIa transport pathway.

Amino Acid Substitution↗

PubMed2002

Identification of functionally important sites in the first intracellular loop of the NaPi-IIa cotransporter.

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 conferring substrate affinity, other mutations at this site were made. Only two mutants yielded significant (32)P(i) uptake and inward P(i)-induced currents with decreased P(i) affinity; for the others, P(i) application suppressed only the Na(+) leak. We suggest that ICL-1 and ECL-3 sites contribute to the transport pathway and that site N199 is implicated in defining the transport mode.

Algorithms↗

PubMed2002

The renal type IIa Na/Pi cotransporter: structure-function relationships.

The type IIa Na/Pi cotransporter mediates proximal tubular brush-border membrane secondary active phosphate (Pi) flux. It is rate limiting in tubular Pi reabsorption and, thus, a final target in many physiological and pathophysiological situations of altered renal Pi handling. In the present short review, we will briefly summarize our current knowledge about the transport mechanism (cycle) as well as particular regions of the transporter protein ("molecular domains") that potentially determine transport characteristics.

Amino Acid Sequence↗