The non-lysosomal β-glucosidase GBA2 is a non-integral membrane-associated protein at the Endoplasmic Reticulum (ER) and golgi.

GBA1 and GBA2 are both beta-glucosidases, which cleave glucosylceramide (GlcCer) to glucose and ceramide. GlcCer is a main precursor for higher order glycosphingolipids but might also serve as intracellular messenger. Mutations in the lysosomal GBA1 underlie Gaucher disease, the most common lysosomal storage disease in humans. Knocking out the non-lysosomal GBA2 in mice results in accumulation of GlcCer outside the lysosomes in various tissues (e. g. testis and liver) and impairs sperm development and liver regeneration. However, the underlying mechanisms are not well understood. To reveal the physiological function of GBA2 and, thereby, of the non-lysosomal GlcCer pool, it is important to characterize the localization of GBA2 and its activity in different tissues. Thus, we generated GBA2-specific antibodies and developed an assay that discriminates between GBA1 and GBA2 without the use of detergent. We show that GBA2 is not, as previously thought, an integral membrane protein but rather a cytosolic protein that tightly associates with cellular membranes. The interaction with the membrane, in particular with phospholipids, is important for its activity. GBA2 is localized at the ER and Golgi, which puts GBA2 in a key position for a lysosome-independent route of GlcCer-dependent signaling. Furthermore, our results suggest that GBA2 might affect the phenotype of Gaucher disease, because GBA2 activity is reduced in Gba1 knock-out fibroblasts and fibroblasts from a Gaucher patient. Our results provide the basis to understand the mechanism for GBA2 function in vivo and might help to unravel the role of GBA2 during pathogenesis of Gaucher disease

Structural insights into conformational changes of a cyclic nucleotide-binding domain in solution from Mesorhizobium loti K1 channel

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site

Importance of the CNGA4 channel gene for odor discrimination and adaptation in behaving mice

Odor stimulation of olfactory sensory neurons (OSNs) leads to both the activation and subsequent desensitization of a heteromultimeric cyclic-nucleotide-gated (CNG) channel present in these cells. The native olfactory CNG channel consists of three distinct subunits: CNGA2, CNGA4, and CNGB1b. Mice in which the CNGA4 gene has been deleted display defective Ca(2+)/calmodulin-dependent inhibition of the CNG channel, resulting in a striking reduction in adaptation of the odor-induced electrophysiological response in the OSNs. These mutants therefore afford an excellent opportunity to assess the importance of Ca(2+)-mediated CNG channel desensitization for odor discrimination and adaptation in behaving animals. By using an operant conditioning paradigm, we show that CNGA4-null mice are profoundly impaired in the detection and discrimination of olfactory stimuli in the presence of an adapting background odor. The extent of this impairment depends on both the concentration and the molecular identity of the adapting stimulus. Thus, Ca(2+)-dependent desensitization of the odor response in the OSNs mediated by the CNGA4 subunit is essential for normal odor sensation and adaptation of freely behaving mice, preventing saturation of the olfactory signal transduction machinery and extending the range of odor detection and discrimination