Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels

Molecular determinants of ion channel tetramerization are well characterized, but those involved in heteromeric channel assembly are less clearly understood. The heteromeric composition of native channels is often precisely controlled. Cyclic nucleotide-gated (CNG) channels from rod photoreceptors exhibit a 3:1 stoichiometry of CNGA1 and CNGB1 subunits that tunes the channels for their specialized role in phototransduction. Here we show, using electrophysiology, fluorescence, biochemistry, and X-ray crystallography, that the mechanism for this controlled assembly is the formation of a parallel 3-helix coiled-coil domain of the carboxy-terminal leucine zipper region of CNGA1 subunits, constraining the channel to contain three CNGA1 subunits, followed by preferential incorporation of a single CNGB1 subunit. Deletion of the carboxy-terminal leucine zipper domain relaxed the constraint and permitted multiple CNGB1 subunits in the channel. The X-ray crystal structures of the parallel 3-helix coiled-coil domains of CNGA1 and CNGA3 subunits were similar, suggesting that a similar mechanism controls the stoichiometry of cone CNG channels

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

The glutamic acid-rich protein-2 (GARP2) is a high affinity rod photoreceptor phosphodiesterase (PDE6)-binding protein that modulates its catalytic properties.

Abstract The glutamic acid-rich protein-2 (GARP2) is a splice variant of the β-subunit of the cGMP-gated ion channel of rod photoreceptors. GARP2 is believed to interact with several membrane-associated phototransduction proteins in rod photoreceptors. In this study, we demonstrated that GARP2 is a high affinity PDE6-binding protein and that PDE6 co-purifies with GARP2 during several stages of chromatographic purification. We found that hydrophobic interaction chromatography succeeds in quantitatively separating GARP2 from the PDE6 holoenzyme. Furthermore, the 17-kDa prenyl-binding protein, abundant in retinal cells, selectively released PDE6 (but not GARP2) from rod outer segment membranes, demonstrating the specificity of the interaction between GARP2 and PDE6. Purified GARP2 was able to suppress 80% of the basal activity of the nonactivated, membrane-bound PDE6 holoenzyme at concentrations equivalent to its endogenous concentration in rod outer segment membranes. However, GARP2 was unable to reverse the transducin activation of PDE6 (in contrast to a previous study) nor did it significantly alter catalysis of the fully activated PDE6 catalytic dimer. The high binding affinity of GARP2 for PDE6 and its ability to regulate PDE6 activity in its dark-adapted state suggest a novel role for GARP2 as a regulator of spontaneous activation of rod PDE6, thereby serving to lower rod photoreceptor dark noise and allowing these sensory cells to operate at the single photon detection limit