Catalytically inactive carbonic anhydrase-related proteins enhance transport of lactate by MCT1

Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to protons and bicarbonate and thereby play a fundamental role in the epithelial acid/base transport mechanisms serving fluid secretion and absorption for whole-body acid/base regulation. The three carbonic anhydrase-related proteins (CARPs) VIII, X, and XI, however, are catalytically inactive. Previous work has shown that some CA isoforms noncatalytically enhance lactate transport through various monocarboxylate transporters (MCT). Therefore, we examined whether the catalytically inactive CARPs play a role in lactate transport. Here, we report that CARP VIII, X, and XI enhance transport activity of the MCT MCT1 when coexpressed in Xenopus oocytes, as evidenced by the rate of rise in intracellular H+ concentration detected using ion-sensitive microelectrodes. Based on previous studies, we suggest that CARPs may function as a 'proton antenna' for MCT1, to drive proton-coupled lactate transport across the cell membrane

Temporal behaviour of cellular H+ buffering

The [H+] is a potent modulator of several physiological processes. Thus, the regulation of cytosolic [H+] is of central importance for the cell and the pH has to be maintained in a narrow range. One fundamental mechanism to maintain pH, besides the transport of acid/base equivalents across the cell membrane, is the buffering of cytosolic H+. The degree to which protons are buffered is quantified by the buffer capacity (or buffer strength or power). M. Koppel & K. Spiro, (Biochem. Zeitschr. 65, 1914) and D.D. van Slyke, (J. Biol. Chem. 52, 1922) independently defined the buffer capacity β as the additive inverse of the differential quotient given by the infinitesimal amount of acid S divided by the actual infinitesimal change in pH:β=-dS/dpH. Until now, the definition was only used for steady state buffering (instantaneous buffering assumed for the intrinsic buffering). In the case that the buffering is not instantaneous (for the CO2/HCO3- buffer system), two different buffer capacities can be defined: βdyn for the dynamic reaction system and βeq for the corresponding equilibrated system. While the last value corresponds to the classical buffer capacity, the last one is by itself less informative. More interesting is its rate of change with respect to time, given by the time derivative β’dyn=dβdyn/dt , which can be seen a quantitative measure how fast the buffering occurs under non-steady-state conditions. We will show in Xenopus laevis oocytes that β’dyn can be modulated without effecting βeq. Such a modulator is e.g. carbonic anhydrase II (CAII) in the presence of the CO2/HCO3- buffer system

Real 3D stochastic particle (H+) simulations

The control and regulation of the intracellular pH is of pivot-al importance for every living organism. To keep the pHi at a constant level, the cell can bind or release protons from molecular binding sites. This process is called H+ buffering. Buffering was so far often measured in equilibrium, but the dynamic development over time has not been considered. We recently developed a new dynamic buffering concept, which considers both the amplitude and the time course of H+ buffering. To gain more knowledge about the dynamics of proton buffering we set up a mathematical model of the processes involved in H+ buffering. Since we have not found any adequate and efficient tool which allows the in-silico simulation of reaction-diffusion processes in cells with complex three-dimensional geometries, we developed a method which is based on a stochastic approach (in contrast to the common PDE solvers), and which consists of a combination of a Monte Carlo particle simulation and an additional Importance Sampling step, which allows the in-finite reuse of already sampled paths. This additional step makes the method extremely efficient. Simulations can be done by factors faster than real-time. We used this tool to simulate the pH dynamics in an oocyte and in an astro-cyte-like shape of model cell with a highly complex geometry. The computation time was compared with a state of the art partial differential equation (PDE) solver

Bergmann glial AMPA receptors are required for fine motor coordination.

The impact of glial neurotransmitter receptors in vivo is still elusive. In the cerebellum, Bergmann glial (BG) cells express alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) composed exclusively of GluA1 and/or GluA4 subunits. With the use of conditional gene inactivation, we found that the majority of cerebellar GluA1/A4-type AMPARs are expressed in BG cells. In young mice, deletion of BG AMPARs resulted in retraction of glial appendages from Purkinje cell (PC) synapses, increased amplitude and duration of evoked PC currents, and a delayed formation of glutamatergic synapses. In adult mice, AMPAR inactivation also caused retraction of glial processes. The physiological and structural changes were accompanied by behavioral impairments in fine motor coordination. Thus, BG AMPARs are essential to optimize synaptic integration and cerebellar output function throughout life