Research Report 2010 - MDC

Determinants of anion-proton coupling inmammalian endosomal CLCsAnselm Zdebik, Eun-Yeong Bergsdorf, Michael Pusch,Giovanni ZifarelliSimilar to the bacterial EcClC-1 protein, and in contrastto their previous classification as Cl - -channels, ClC-4and ClC-5 are antiporters that exchange chloride forprotons. By mutagenesis and biophysical analysis, weshowed that a glutamate at the cytoplasmic face of thetransporter is important for proton coupling. It apparentlyserves as a proton acceptor. Cl - /H + -transport persistswhen this residue is mutated to other titratableamino-acids, but transport is altogether stopped whenit is mutated e.g. to alanine. Transport is restored whena centrally located ‘gating glutamate’ is neutralized,which eliminates Cl/proton coupling at a centralexchange site and therefore eliminates the need of protonsfor Cl transport.Residues important for nitrate transport in plantand mammalian CLC proteinsEun-Yeong Bergsdorf, Anselm ZdebikAtClC-a from the plant Arabidopsis has been shown byBarbier-Brygoo and colleagues to function as a NO3 - /H +exchanger with which plants accumulate the nutrientnitrate into their vacuoles. This requires tightly coupledcountertransport. However, when nitrate is transportedby mammalian endosomal ClC-4 and ClC-5, it is largelyuncoupled from protons. Sequence comparison identifieda serine in a highly conserved signature sequenceof ClC-4/5 that is exchanged by a proline in AtClC-a.Mutating the serine of ClC-5 to proline led to tightlycoupled nitrate/proton exchange, whereas the planttransporter lost its preference for nitrate when its prolinewas mutated to serine. The ClC-0 Cl channel fromelectric fish also gained nitrate selectivity when thisproline was introduced. We have thus identified aresidue crucial for anion selectivity and have also beenable for the first time to functionally express a plantCLC in animal cells.KCl and NaK2Cl cotransportersCarsten Pfeffer, Guillermo Spitzmaul, Patricia Seja,Valentin Stein, Hannes MaierWe have previously knocked-out all KCl-cotransporterisoforms (KCC1-4) in mice which led to specific andhighly interesting phenotypes. We are continuing ourstudies on KCCs with conditional KOs. Our major focusis on KCCs expressed in neurons, where KCC2 in particularlowers the cytoplasmic chloride concentration.Such a low concentration is necessary for the inhibitoryaction of GABA and glycine, which act on ligand-gatedchloride channels. We are currently investigating variousmouse lines in which KCC2 has been inactivated inspecific sets of neurons.We have also studied the transporter that is the majorplayer in elevating cytoplasmic chloride in neuronsbefore the expression of KCC2 kicks in, namely theNaK2Cl cotransporter NKCC1. We found that NKCC1 isan important, though not the only, transporter elevatingintraneuronal chloride. The excitability and spontaneousnetwork activity of hippocampal slices werereduced in the first 10 days after birth. This correlatedwith a delay in synapse maturation. However, in contrastto speculations by others, no morphologicalchanges were observed.KCNQ potassium channelsMatthias Heidenreich, Guillermo Spitzmaul, VityaVerdanyan, Pawel FidzinskiThere are five different isoforms of KCNQ (Kv7) potassiumchannels, KCNQ1- KCNQ5. KCNQ2-KCNQ5 mediate‘M-currents’ that regulate neuronal excitability. We hadpreviously shown that KCNQ2 and KCNQ3 underlie aform of human epilepsy and that dominant KCNQ4mutations are a cause of human deafness and havepublished a few years ago a mouse model for KCNQ4deafness. We are now investigating possible vestibularphenotypes in these mice and are generating othermouse models.Selected PublicationsBlanz J., Schweizer M., Auberson M., Maier H., Muenscher A., Hübner C.A.,Jentsch T.J. (2007). Leukoencephalopathy upon disruption of the chloridechannel ClC-2. J. Neurosci. 27, 6581-6589.Maritzen T., Keating D.J., Neagoe I., Zdebik A.A., Jentsch T.J. (2008). Role ofthe vesicular chloride transporter ClC-3 in neuroendocrine tissue. J.Neurosci. 28, 10587-10598.Rickheit G., Maier H., Strenzke N., Andreescu C.E., De Zeeuw C.I., ZdebikA.A., Jentsch T.J. (2008). Endocochlear potential depends on chloridechannels: mechanism underlying deafness in Bartter syndrome IV. EMBOJ. 27, 2907-2917.Pfeffer C.K., Stein V., Keating D.J., Maier H., Rinke I., Rudhard Y., HentschkeM., Rune G., Jentsch T.J., Hübner C.A. (2009). NKCC1-Dependent GABAergicExcitation Drives Synaptic Network Maturation During EarlyHippocampal Development. J. Neurosci. 29, 3419-3430.Bergsdorf, E.-Y., Zdebik A.A., Jentsch T.J. (2009). Residues important fornitrate/proton coupling in plant and mammalian CLC transporters. J. Biol.Chem. 284, 11184-11193.Function and Dysfunction of the Nervous System 157