Characterization of voltage-sensing phosphatase regulation on membrane phosphoinositides and voltage-gated calcium channels

Abstract

After Luigi Galvani’s first observation in 1789, it has been found that bioelectricity plays crucial roles in biological systems. Most vital activities including sensory reception, decision making, learning, memory, cognition, motion, and generation of cardiac impulse are electrically mediated in precise and works of many ion channels and their regulators in concert. For instance, in excitable cells, external stimulations such as light, pressure, temperature, pH change, and ligand can activate ion channels and evoke membrane depolarization. If the stimulations are sufficiently strong to overcome threshold, action potential is generated and propagates by sequential activation of voltage-gated sodium channel (NaV) and voltage-gated potassium channel (KV). Then, the action potential activates voltage-gated calcium channels (CaV) in presynaptic terminal and triggers secretion of neuro-transmitter, resulting in synaptic transmission to post synaptic neuron. Regulators of ion channels precisely control shape and strength of the signal at each step. Phosphoinositides (PIs) are phosphorylated isomers of phosphoinositol (PI), which has inositol head group and fatty acid tail group, and are named after phosphorylation at 3, 4, or 5 position of the inositol ring. They are minor (<1%) lipid in cell membranes and dynamically and accurately maintained by working of phosphoinositide kinases and phosphatases. Also, they regulates important cellular and physiological events such as cell differentiation, migration, cell-shaping, axon-regeneration, cell death / survival, tumorigenesis, protein regulation, fertilization and so on. Additionally, discovery and development of molecular probes attached with fluorescence proteins enables us to observe real-time concentration of PIs in live cells. Among these PIs, role of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is relatively well established. Not only as precursor of IP3 which induces release of Ca2+ from endoplasmic reticulum (ER), PI(4,5)P2 is known to directly regulate various ion channels including CaV channels, one of our study targets. Recently cloned voltage-sensing phosphatases (VSPs) extend the concept of bioelectrical signal and their application. VSPs are transmembrane proteins that have voltage-sensing domain mimicking that of voltage-gated ion channels, and cytosolic catalytic domain homologous to phosphatase and tensin homolog (PTEN), a phosphoinositide PI(3,4)P2 and PI(3,4,5)P3 3-phosphatase. Thus, propagation of electricity can be transduced into catalytic activity. However, unlike PTEN, VSPs have a wider range of substrates, cleaving 3-phosphate from PI(3,4)P2 and probably PI(3,4,5)P3 and 5-phosphate from PI(4,5)P2, and PI(3,4,5)P3. Recent proposals say these reactions have differing voltage dependence. Using FRET probes specific for different phosphoinositides in living cells with zebrafish VSP, we quantitate both voltagedependent 5- and 3-phosphatase subreactions against endogenous substrates. These activities become apparent with different voltage thresholds, voltage sensitivities, and catalytic rates. As an analytical tool, we refine a kinetic model that includes the endogenous pools of phosphoinositides, endogenous phosphatase and kinase reactions connecting them, and four exogenous voltage-dependent 5- and 3- phosphatase subreactions of VSP. We show that apparent voltage threshold differences for seeing effects of the 5- and 3-phosphatase activities in cells are not due to different intrinsic voltage dependence of these reactions. Rather the reactions have a common voltage dependence, and apparent differences arise only because each VSP subreaction has a different absolute catalytic rate that begins to surpass the respective endogenous enzyme activities at different voltages. For zebrafish VSP, our modeling also revealed clear 3-phosphatase activity against PI(3,4,5)P3, but it is 55-fold slower than 5-phosphatase activity against PI(4,5)P2, thus PI(4,5)P2, generated more slowly from dephosphorylating PI(3,4,5)P3, might never accumulate. When 5-phosphatase activity was counteracted by coexpression of a phosphatidylinositol 4- phosphate 5-kinase, there was accumulation of PI(4,5)P2 in parallel to PI(3,4,5)P3 dephosphorylation, demonstrating that VSPs also cleave the 3-phosphate of PI(3,4,5)P3. Using these catalytic properties of Dr-VSP, role of CaV β subunits on determining portion of two inhibition pathways by Gq-protein coupled receptor (GqPCR) activation was investigated. GPCRs signal through molecular messengers, such as Gβγ, Ca2+, and PI(4,5)P2, to modulate N-type CaV2.2 channels, playing a crucial role in regulating synaptic transmission. In our pervious studies, activation of Dr-VSP always showed weaker response than activation of M1 muscarinic receptor in CaV2.2 current inhibition (10% and 40%, respectively) although either are known as PI(4,5)P2 dependent pathways. In this study, we firstly identified that Gβγ dissociated from M1 GqPCR further inhibited CaV current beside PI(4,5)P2 depletion. We also report that the location of CaV β subunits is key to determining the voltage dependence of CaV2.2 channel modulation by GqPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M1 receptors, together with CaV N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for CaV2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of CaV2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of CaV2.2 current after M1 receptor activation was markedly reduced and delayed, whereas the delay between PIP2 hydrolysis and inhibition of CaV2.2 current was decreased. When the Gβγ-insensitive CaV2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M1 receptors act through Gβγ to inhibit CaV2.2 channels bearing membranelocalized CaV β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylationnegative mutant β2a(C3,4S), reduced the voltage dependence of M1 muscarinic inhibition of CaV2.2 channels, whereas it increased inhibition mediated by PIP2 depletion. Together, our results indicate that, with membrane-localized CaV β subunits, CaV2.2 channels are subject to Gβγ-mediated voltagedependent inhibition, whereas cytosol-localized β subunits confer more effective PIP2-mediated voltageindependent regulation. Thus, the voltage dependence of GqPCR regulation of calcium channels can be determined by the location of isotype-specific CaV β subunits. These results suggest novel physiological roles of VSPs and CaV channels. Since VSPs also dephosphorylate 3-phosphate of PI(3,4,5)P3 as PTEN does, VSPs may participate in axon re-generation, spermato-genesis or stem cell differentiation which might even be triggered and regulated by electrochemical signals. VSPs also can compensate defect of PTEN activity in neural development or tumor suppression. Different temporal property of voltage-dependent and -independent inhibitions on CaV channels may be resposible for formation of M1R induced long term potentialtion and depression. ⓒ 2016 DGIST