The Na+/Ca2+, K+ exchanger NCKX4 is required for efficient cone-mediated vision

Calcium (Ca2+) plays an important role in the function and health of neurons. In vertebrate cone photoreceptors, Ca2+ controls photoresponse sensitivity, kinetics, and light adaptation. Despite the critical role of Ca2+ in supporting the function and survival of cones, the mechanism for its extrusion from cone outer segments is not well understood. Here, we show that the Na+/Ca2+, K+ exchanger NCKX4 is expressed in zebrafish, mouse, and primate cones. Functional analysis of NCKX4-deficient mouse cones revealed that this exchanger is essential for the wide operating range and high temporal resolution of cone-mediated vision. We show that NCKX4 shapes the cone photoresponse together with the cone-specific NCKX2: NCKX4 acts early to limit response amplitude, while NCKX2 acts late to further accelerate response recovery. The regulation of Ca2+ by NCKX4 in cones is a novel mechanism that supports their ability to function as daytime photoreceptors and promotes their survival

Engineering the folding pathway of insect cells: Generation of a stably transformed insect cell line showing improved folding of a recombinant membrane protein

he baculovirus-insect cell expression system has proven to be a valuable tool for the high level production of a multitude of recombinant proteins. However, production of membrane proteins in infected insect cells is often hampered by incorrect folding and processing which results in the accumulation of non-functional protein. Here, we report the construction of a Sf9 insect cell line stably transformed with the 12 ninaA gene from D. melanogaster rhabdomere. Engineered Sfn insect cells infected with a recombinant baculovirus bearing the human dopamine transporter gene under the control of the polyhedrin promoter showed a ≤ 5 times enhanced uptake of [3H]dopamine in comparison to similarly infected Sf9 cells. This increase in specific transport activity was not due to an altered K(m) value in the Sfn cell line. The uptake in infected Sfn cells was blocked by the peptidyl-prolyl cis/trans isomerase inhibitor cyclosporin A which had no effect on infected Sf9 cells. From these results we conclude that the prolyl-cis/trans isomerase activity of the ninaA in the stably transformed Sfn cell line was responsible, directly or indirectly, for the improved folding of the heterologously produced human dopamine transporter

Production of G protein-coupled receptors in yeast

Yeasts combine the advantages of fast and easy handling with the potential to perform eukaryotic post-translational modifications and are for this reason interesting hosts for heterologous production of G-protein-coupled receptors. The possibility to connect foreign receptors to a yeast internal MAP kinase pathway was used to establish yeast-based systems for high-throughput screening of compound libraries. In addition, yeasts have the potential for high level production of G-protein-coupled receptors. In this field, non-Saccharomyces yeasts seems to be interesting alternatives to S. cerevisiae, as well as to systems based on higher eukaryotic cells

Production of G-protein-coupled receptors in yeast.

Yeasts combine the advantages of fast and easy handling with the potential to perform eukaryotic post-translational modifications and are for this reason interesting hosts for heterologous production of G-protein-coupled receptors. The possibility to connect foreign receptors to a yeast internal MAP kinase pathway was used to establish yeast-based systems for high-throughput screening of compound libraries. In addition, yeasts have the potential for high level production of G-protein-coupled receptors. In this field, non-Saccharomyces yeasts seems to be interesting alternatives to S. cerevisiae, as well as to systems based on higher eukaryotic cells

Expression of an Integral Membrane Protein, the 5HT_5A Receptor

Research in the field of membrane proteins has undergone explosive growth during the last decade, primarily owing to the influence of the powerful techniques of modern molecular biology. Membrane proteins fulfill essential functions, such as communication, selective transport of metabolites and ions, and energy transformation. It is estimated that one-third of the genes of an organism encode integral membrane proteins (1). We are just now beginning to understand the molecular structures of this group of proteins and how they function within the confines of the cellular membranes. Among the different families of membrane proteins, the so-called G protein-coupled receptors (GPCRs) comprise the largest family. From the viewpoint of pharmacology, this family is of great importance, since about 60% of all pharmaceuticals known today mediate their effects via interaction with GPCRs. Therefore, much progress has been made in the characterization of the pharmacological and biochemical properties, as well as the signal transduction mechanisms of the GPCRs. Nevertheless, in order to understand the function and molecular dynamics of these receptors, detailed structural information will be needed. Despite the steady progress in understanding of GPCRs, solid three-dimensional (3D) structural data are still missing. To date, the crystallization and 3D determination have been successfully performed on only a handful of membrane proteins. All these structural determinations were performed on membrane proteins that are naturally highly expressed and can be purified in large quantities from their natural sources

A Rapid Method for Amplification of Plastome DNA-Fragments from Spinacia oleracea by PCR

Polymerase Chain Reaction (PCR) was used as a very powerful technique to amplify DNA-fragments. Isolated chloroplasts and a crude suspension of small leaf pieces of Spinacia oleracea were osmotically shocked and subsequently heated. Aliquots of the resulting homogenate including the organelle DNA were directly added to basic reaction mixtures to carry out PCR without additional isolation- or purification steps. A 180 by DNA-fragment from the psbA-gene was amplified in both cases. In parallel, plasmid-DNA (pHT1) bearing the psbA-gene was subjected to PCR retaining unchanged reaction conditions. Identical DNA-fragments were obtained, as the following sequencing revealed. With the method described one can amplify any known plastome-fragment in a few hour