Owen T. Jones

Voltage-gated calcium channels (Ca_v) exist as heteromultimers comprising a pore-forming α₁ with accessory β and α₂δ subunits which modify channel trafficking and function. We previously showed that α₂δ-1 (and likely the other mammalian α₂δ isoforms - α₂δ-2, 3 and 4) is required for targeting Ca_vs to lipid rafts, although the mechanism remains unclear. Whilst originally understood to have a classical type I transmembrane (TM) topology, recent evidence suggests the α₂δ subunit contains a glycosylphosphatidylinositol (GPI)-anchor that mediates its association with lipid rafts. To test this notion, we have used a strategy based on the expression of chimera, where the reported GPI-anchoring sequences in the gabapentinoid-sensitive α₂δ-1 subunit have been substituted with those of a functionally inert Type I TM-spanning protein – PIN-G. Using imaging, electrophysiology and biochemistry, we find that lipid raft association of PIN-α₂δ is unaffected by substitution of the GPI motif with the TM domain of PIN-G. Moreover, the presence of the GPI motif alone is not sufficient for raft localisation, suggesting that upstream residues are required. GPI-anchoring is susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage. However, whilst raft localisation of PIN-α₂δ is disrupted by PI-PLC treatment, this is assay-dependent and non-specific effects of PI-PLC are observed on the distribution of the endogenous raft marker, caveolin, but not flotillin. Taken together, these data are most consistent with a model where α₂δ-1 retains its type I transmembrane topology and its targeting to lipid rafts is governed by sequences upstream of the putative GPI anchor, that promote protein-protein, rather than lipid-lipid interactions.

The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones.

The last decade has witnessed an exponential increase in interest in one of the great mysteries of nerve cell biology: Specifically, how do neurons know where to place the ion channels that control their excitability? Many of the most important insights have been gleaned from studies on the voltage-gated potassium channels (Kvs) which underlie the shape, duration and frequency of action potentials. In this review, we gather recent evidence on the expression, trafficking and maintenance mechanisms which control the surface density of Kvs in different subcellular compartments of neurons and how these may be regulated to control cell excitability.

APS (Acta Pharmacologica Sinica), the top pharmacology research journal based in China, publishes original articles and reviews on all aspects of pharmacology and the related life sciences

Background The identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. Unfortunately, the analysis of trafficking signals is complicated by their topography, hierarchical nature and regulation. Powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. However, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells. Results Consequently, we have engineered a mammalian expression vector encoding a novel trafficking reporter – pIN-G – consisting of a simple, type I integral protein bearing permissive intra/extracellular cloning sites, green fluorescent protein (GFP), cMyc and HA epitope tags. Fluorescence imaging, flow cytometry and biochemical assays of transfected HEK293 cells, confirm the size, topology and surface expression of PIN-G. Moreover, a pIN-G fusion construct, containing a Trans-Golgi Network (TGN) targeting determinant, internalises rapidly from the cell surface and localises to the TGN. Additionally, another PIN-G fusion protein and its mutants reveal trafficking determinants in the cytoplasmic carboxy terminus of Kv1.4 voltage-gated potassium channels. Conclusion Together, these data indicate that pIN-G is a versatile, powerful, new reporter for analysing signals controlling membrane protein trafficking, surface expression and dynamics.