PI(4,5)P-2 and L-type Ca2+ Channels Partner Up to Fine-Tune Ca2+ Dynamics in beta Cells

The PI(4,5)P2 level in the plasma membrane is dynamically regulated by cytoplasmic ATP production and receptor-mediated transmembrane signaling cascades. In this issue of Cell Chemical Biology, Xie et al. (2016) use optogenetics to micro-manipulate membrane PI(4,5)P2 and reveal how acute PI(4,5)P2 changes can alter intracellular Ca2+ dynamics and insulin secretion in pancreatic β cells. © 2016 Elsevier Ltd

Membrane-localized beta-subunits alter the PIP2 regulation of high-voltage activated Ca2+ channels

The β-subunits of voltage-gated Ca 2+ (Ca V) channels regulate the functional expression and several biophysical properties of high-voltage-activated Ca V channels. We find that Ca V β-subunits also determine channel regulation by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP 2). When Ca V1.3, -2.1, or -2.2 channels are cotransfected with the β3-subunit, a cytosolic protein, they can be inhibited by activating a voltage-sensitive lipid phosphatase to deplete PIP 2. When these channels are coexpressed with a β2a-subunit, a palmitoylated peripheral membrane protein, the inhibition is much smaller. PIP 2 sensitivity could be increased by disabling the two palmitoylation sites in the β2a-subunit. To further test effects of membrane targeting of Ca V β-subunits on PIP 2 regulation, the N terminus of Lyn was ligated onto the cytosolic β3-subunit to confer lipidation. This chimera, like the Ca V β2a-subunit, displayed plasma membrane localization, slowed the inactivation of Ca V2.2 channels, and increased the current density. In addition, the Lyn-β3 subunit significantly decreased Ca Vchannel inhibition by PIP 2 depletion. Evidently lipidation and membrane anchoring of Ca V β-subunits compete with the PIP 2 regulation of high-voltage-activated Ca V channels. Compared with expression with Ca V β3-subunits alone, inhibition of Ca V2.2 channels by PIP 2 depletion could be significantly attenuated when β2a was coexpressed with β3. Our data suggest that the Ca V currents in neurons would be regulated by membrane PIP 2 to a degree that depends on their endogenous β-subunit combinations.

Analysis of Phosphoinositide-Binding Properties and Subcellular Localization of GFP-Fusion Proteins

Specific protein-phosphoinositide (PI) interactions are known to play a key role in the targeting of proteins to specific cellular membranes. Investigation of these interactions would be greatly facilitated if GFP-fusion proteins expressed in mammalian cells and used for their subcellular localization could also be employed for in vitro lipid binding. In this study, we found that lysates of cells overexpressing GFP-fusion proteins could be used for in vitro protein-PI binding assays. We applied this approach to examine the PI-binding properties of Aplysia Sec7 protein (ApSec7) and its isoform ApSec7(VPKIS), in which a VPKIS sequence is inserted into the PH domain of ApSec7. EGFP-ApSec7 but not EGFP-ApSec7(VPKIS) did specifically bind to PI(3,4,5)P3 in an in vitro lipid-coated bead assay. Overexpression of EGFP-ApSec7 but not EGFP-ApSec7(VPKIS) did induce neurite outgrowth in Aplysia sensory neurons. Structure modeling analysis revealed that the inserted VPKIS caused misfolding around the PI(3,4,5)P3-binding pocket of ApSec7 and disturbed the binding of PI(3,4,5)P3 to the pleckstrin homology (PH) domain. Our data indicate that plasma membrane localization of EGFP-ApSec7 via the interaction between its PH domain and PI(3,4,5)P3 might play a key role in neurite outgrowth in Aplysia. © 2015 AOCS.