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Physiology and pathophysiology of membrane transport processes

Ion channels and transporters are crucial for the generation, propagation and transmission of electrical signals. Since the lipid bilayer of biological membrane in impermeant for polar and charged molecules, channels and transporters are also necessary for the transmembrane transport of nutrients, electrolytes and signal molecules.
There are various human pathological conditions that are caused by genetic or acquired alterations of ion channels and transporters. Studying the pathophysiology of these diseases can provide insights into previously unknown cellular functions of such proteins. Since ion channels and transporters represent attractive targets for pharmacological modification, such studies might also contribute to the development of novel therapeutic approaches.
Our research focuses on the physiology and pathophysiology of membrane transport. We aim at understanding of molecular mechanisms underlying ion channel and transporter function or dysfunction and of the consequences of altered membrane transport in human organs. We use a variety of experimental and theoretical approaches. Electrophysiological techniques such as patch clamp or microelectrode voltage-clamp recordings, fluorescent indicators or radiotracer flux measurements are used to quantify transport functions. Conformational changes underlying channel and transporter functions are addressed using fluorescence spectroscopy. Moreover, we have recently started to use computational approaches to define the atomistic basis of ion permeation. To define sequence determinants of particular protein functions we express channels and transporters – either with normal or altered protein sequence - in mammalian cells or oocytes. Another approach is expression of proteins in bacteria, followed by purification, reconstitution and functional analysis. To study the consequences of particular transport functions in native cells we record from dissociated neurons/glial cells or from slice preparations. We have furthermore mouse models diseases generated to study the pathophysiology of selected monogenetic diseases.
At present we mainly concentrate on three classes of membrane proteins, ClC chloride channels and transporters, the SLC26 family of multifunctional anion exchangers and two classes of glutamate transporters. All these proteins are important for distinct cellular functions. Their physiological impact is underlined by the existence of monogenetic diseases caused by mutations in genes encoding these proteins.

 

Abb. 1Human diseas associated with dysfunctional ion channels or transporters

Glutamate transporters in episodic ataxia and epilepsy


Episodic ataxias are rare human conditions that are characterized by paroxysmal motor incoordination. We are interested in one form of episodic ataxia that is caused by mutations in the gene (SLC1A3) that encodes the glial glutamate transporter EAAT1. All EAAT glutamate transporters are dual function transport proteins that are not only secondary active glutamate transporters, but also anion channels. We recently demonstrated that a naturally occuring mutation - that predicts the exchange of a highly conserved proline by arginine and that was found in a patient with ataxia as well as with epilepsy - has opposite effects on these two transport functions, P290R reduces the EAAT1 glutamate transport and dramatically enhances the EAAT1 anion current.
We are studying the molecular mechanisms of P290R-induced alterations of EAAT1 transport functions. To understand how changes in EAAT1 anion currents can result in neuronal hyperexcitability we have a P290R knock-in mouse model generated that we are currently analysing with electrophysiological and microscopic techniques.

P290R knock in mouse exhibit pronounced ataxia and epilepsy P290R results in a pronounced gain-of-function of EAAT1 anion currents in heterologous expression systems. This figure shows whole cell patch clamp recordings of chloride currents from HEK293T cells expressing WT or P290R hEAAT1 cells.

Ion channels in the regulation of blood pressure

The urinary excretion of electrolytes and fluid is an important regulator of blood pressure. Changes in renal electrolyte reabsorption can cause severe disturbances of human blood pressure. These pathological conditions can be caused by ion channel dysfunction in the nephron or in changes in electrical properties of cells that produce and release hormones regulation renal function.
Anion channels in the thin and thick ascending limb of Henle are crucial for normal urinary concentration. Such channels consist of two pore-forming ClC-K subunits and a so far unknown number of the accessory subunit barttin. ClC-K channels are only functional when co-expressed with barttin. Mutations in the genes encoding ClC-Ka (CLCNKA), ClC-Kb (CLCNKB) or barttin (BSND) disturb blood pressure regulation and can cause genetic forms of deafness. We study the regulation of ClC-K by barttin using a combination of molecular biology, protein biochemistry, super-resolution microscopy and computational biology. We expect novel insights about novel potential pharmacological approaches for the treatment of high blood pressure and other cardio-vascular conditions from this work.

We recently became interested into a rather frequently occurring form of hypertension that is caused by increased levels of the mineral-corticoid aldosterone and that results from genetic alterations in calcium channels. We study the functional consequences of calcium channel mutations in hyper-aldosteronism and the cellular roles of WT and mutant channels.

Abb. 3Physiological functions of ClC-K channels in the thin and thick ascending limb of Henle and the stria vascularis of the inner ear.

Transport processes in synaptic vesicles


Neurons communicate via chemical synapses with each other. Such synapses are of special importance for complex cognitive processes in the central nervous system since they adjust their function in response to alteration in synaptic and neuronal activity.
Synaptic transmission is initiated by the release of neurotransmitters via fusion of synaptic vesicles with the plasma membrane of presynaptic nerve terminals. An important parameter determining the efficacy of synaptic transmission is the concentration of neurotransmitter in synaptic vesicles. We investigate the mechanisms of neurotransmitter accumulation in synaptic vesicles.

Abb. 4Neurotransmitters are accumulated in synaptic vesicles by secondary-active neurotransmitter transporters that utilize the electrochemical H+ gradient across the vesicular membrane

Selected publications

Ewers, D., Becher, T., Machtens, J. P., Weyand, I., and Fahlke, Ch. (2013) Induced fit substrate binding to an archeal glutamate transporter homologue. Proc Natl Acad Sci U S A 110, 12486-12491

Fahlke, Ch., Rüdel, R., Mitrovic, N., Zhou, M., and George, A. L., Jr. (1995) An aspartic acid residue important for voltage-dependent gating of human muscle chloride channels. Neuron 15, 463-472

Fahlke, Ch., Beck, C. L., and George, A. L., Jr. (1997) A mutation in autosomal dominant myotonia congenita affects pore properties of the muscle chloride channel. Proc Natl Acad Sci USA 94, 2729-2734

Fahlke, Ch., Yu, H. T., Beck, C. L., Rhodes, T. H., and George, A. L., Jr. (1997) Pore-forming segments in voltage-gated chloride channels. Nature 390, 529-532

Fischer, M., Janssen, A. G., and Fahlke, Ch. (2010) Barttin activates ClC-K channel function by modulating gating. J Am.Soc.Nephrol. 21, 1281-1289

Gendreau, S., Voswinkel, S., Torres-Salazar, D., Lang, N., Heidtmann, H., Detro-Dassen, S., Schmalzing, G., Hidalgo, P., and Fahlke, Ch. (2004) A trimeric quaternary structure is conserved in bacterial and human glutamate transporters. J Biol.Chem.279, 39505-39512

Melzer, N., Biela, A., and Fahlke, Ch. (2003) Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels. J Biol.Chem. 278, 50112-50119

Riazuddin, S., Anwar, S., Fischer, M., Ahmed, Z. M., Khan, S. Y., Janssen, A. G., Zafar, A. U., Scholl, U., Husnain, T., Belyantseva, I. A., Friedman, P. L., Riazuddin, S., Friedman, T. B., and Fahlke, Ch. (2009) Molecular basis of DFNB73: mutations of BSND can cause nonsyndromic deafness or Bartter syndrome. Am.J.Hum.Genet. 85, 273-280

Scholl, U. I., Goh, G., Stolting, G., de Oliveira, R. C., Choi, M., Overton, J. D., Fonseca, A. L., Korah, R., Starker, L. F., Kunstman, J. W., Prasad, M. L., Hartung, E. A., Mauras, N., Benson, M. R., Brady, T., Shapiro, J. R., Loring, E., Nelson-Williams, C., Libutti, S. K., Mane, S., Hellman, P., Westin, G., Akerstrom, G., Bjorklund, P., Carling, T., Fahlke, Ch., Hidalgo, P., and Lifton, R. P. (2013) Somatic and germline CACNA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism. Nat Genet45, 1050-1054

Scholl, U., Hebeisen, S., Janssen, A. G., Muller-Newen, G., Alekov, A., and Fahlke, Ch. (2006) Barttin modulates trafficking and function of ClC-K channels. Proc.Natl.Acad.Sci.U.S.A 103, 11411-11416

Stölting, G., Teodorescu, G., Begemann, B., Schubert, J., Nabbout, R., Toliat, M. R., Sander, T., Nurnberg, P., Lerche, H., and Fahlke, Ch. (2013) Regulation of ClC-2 gating by intracellular ATP. Pflugers Arch 465, 1423-1437

Winter, N., Kovermann, P., and Fahlke, Ch. (2012) A point mutation associated with episodic ataxia 6 increases glutamate transporter anion currents. Brain 135, 3416-3425

 



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