Nucleoside diphosphate kinase B is required for the formation of heterotrimeric G protein containing caveolae.

Caveolae are flask-shaped invaginations in the plasma membrane that serve to compartmentalize and organize signal transduction processes, including signals mediated by G protein-coupled receptors and heterotrimeric G proteins. Herein we report evidence for a close association of the nucleoside diphosphate kinase B (NDPK B) and caveolin proteins which is required for G protein scaffolding and caveolae formation. A concomitant loss of the proteins NDPK B, caveolin isoforms 1 (Cav1) and 3, and heterotrimeric G proteins occurred when one of these proteins was specifically depleted in zebrafish embryos. Co-immunoprecipitation of Cav1 with the G protein Gβ-subunit and NDPK B from zebrafish lysates corroborated the direct association of these proteins. Similarly, in embryonic fibroblasts from the respective knockout (KO) mice, the membrane content of the Cav1, Gβ, and NDPK B was found to be mutually dependent on one another. A redistribution of Cav1 and Gβ from the caveolae containing fractions of lower density to other membrane compartments with higher density could be detected by means of density gradient fractionation of membranes derived from NDPK A/B KO mouse embryonic fibroblasts (MEFs) and after shRNA-mediated NDPK B knockdown in H10 cardiomyocytes. This redistribution could be visualized by confocal microscopy analysis showing a decrease in the plasma membrane bound Cav1 in NDPK A/B KO cells and vice versa and a decrease in the plasma membrane pool of NDPK B in Cav1 KO cells. Consequently, ultrastructural analysis revealed a reduction of surface caveolae in the NDPK A/B KO cells. To prove that the disturbed subcellular localization of Cav1 in NDPK A/B KO MEFs as well as NDPK B in Cav1 KO MEFs is a result of the loss of NDPK B and Cav1, respectively, we performed rescue experiments. The adenoviral re-expression of NDPK B in NDPK A/B KO MEFs rescued the protein content and the plasma membrane localization of Cav1. The expression of an EGFP-Cav1 fusion protein in Cav1-KO cells induced a restoration of NDPK B expression levels and its appearance at the plasma membrane. We conclude from these findings that NDPK B, heterotrimeric G proteins, and caveolins are mutually dependent on each other for stabile localization and caveolae formation at the plasma membrane. The data point to a disturbed transport of caveolin/G protein/NDPK B complexes from intracellular membrane compartments if one of the components is missing

Functional mapping of GABA A receptor subtypes in the amygdala

The physiological significance of the large diversity of GABA A receptors is poorly understood. Using mice, which carry a point mutation that renders specific subtypes of GABA A receptors diazepam insensitive, it was recently discovered that particular types of GABA A receptors are involved in specific, behaviorally relevant signaling pathways. We have used these mice to study inhibitory synaptic transmission in the amygdala. GABA A receptor-mediated inhibitory postsynaptic currents (IPSCs) per se were not affected by the point mutations. Their modulation by diazepam, however, was altered depending on the genotype of the mice studied. Based on the different responses to diazepam, we found that IPSCs in the lateral/basolateral amygdala were mediated by both alpha2- and alpha1-subunit-containing GABA A receptors whereas those in the central amygdala were mediated only by alpha2-subunit-containing GABA A receptors. Immunohistochemical staining corroborated these findings at a morphological level. To investigate a possible link between interneuron and receptor diversity, we selectively depressed release from the subset of GABAergic terminals carrying type 1 cannabinoid receptors. These receptors are known to modulate amygdala-mediated behavior. Application of a type 1 cannabinoid receptor agonist resulted in a selective reduction of inhibitory current mediated by alpha1-subunit-containing GABA A receptors. Mice with specific diazepam-insensitive GABA A receptor subtypes therefore provide a novel tool to investigate GABA A receptor distribution and the organization of inhibitory circuits at a functional level. The crucial role of the amygdala for the mediation of anxiety is in agreement with the part that alpha2-subunit-containing GABA A receptors play in anxiolysis and their important function in this area of the brain