RESUMEN
Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl- channel activated by intracellular Ca2+ and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P2 degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P2 depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P2 sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current (i), but not of channel number (N) or open probability (PO). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P2, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P2 In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P2-binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P2, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.
Asunto(s)
Empalme Alternativo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Fosfatidilinositoles/metabolismo , Regulación Alostérica , Animales , Anoctamina-1/química , Sitios de Unión , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Ratones , Modelos Moleculares , Conformación Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Isoformas de Proteínas , Relación Estructura-ActividadRESUMEN
ß subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B-ß interactions by rapamycin-translocatable CaV ß subunits that allow drug-induced sequestration and uncoupling of the ß subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified ß subunits, except membrane-tethered ß2a and ß2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable ß subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound ß subunits are very stable. However, the interaction becomes dynamic when other competing ß isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free ß isoforms around the channel reveals a dynamic aspect to the α1B-ß interaction. On the other hand, translocatable ß subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable ß subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B-ß subunits can be overcome by other free ß isoforms, permitting dynamic changes in channel properties in intact cells.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/metabolismo , Activación del Canal Iónico/fisiología , Fosfatidilinositoles/metabolismo , Sirolimus/metabolismo , Animales , Unión Competitiva , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Isoformas de Proteínas , Subunidades de Proteína , Transporte de Proteínas , RatasRESUMEN
G protein-coupled receptors (GPCRs) regulate diverse intracellular signaling pathways through the activation of heterotrimeric G proteins. However, the effects of the sequential activation-deactivation cycle of G protein on the conformational changes of GPCRs remains unknown. By developing a Förster resonance energy transfer (FRET) tool for human M3 muscarinic receptor (hM3R), we find that a single-receptor FRET probe can display the consecutive structural conversion of a receptor by G protein cycle. Our results reveal that the G protein activation evokes a two-step change in the hM3R structure, including the fast step mediated by Gq protein binding and the subsequent slower step mediated by the physical separation of the Gαq and Gßγ subunits. We also find that the separated Gαq-GTP forms a stable complex with the ligand-activated hM3R and phospholipase Cß. In sum, the present study uncovers the real-time conformational dynamics of innate hM3R during the downstream Gq protein cycle.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Proteínas de Unión al GTP , Humanos , Fosfolipasa C betaRESUMEN
The dynamic metabolism of membrane phosphoinositide lipids involves several cellular compartments including the ER, Golgi, and plasma membrane. There are cycles of phosphorylation and dephosphorylation and of synthesis, transfer, and breakdown. The simplified phosphoinositide cycle comprises synthesis of phosphatidylinositol in the ER, transport, and phosphorylation in the Golgi and plasma membranes to generate phosphatidylinositol 4,5-bisphosphate, followed by receptor-stimulated hydrolysis in the plasma membrane and return of the components to the ER for reassembly. Using probes for specific lipid species, we have followed and analyzed the kinetics of several of these events during stimulation of M1 muscarinic receptors coupled to the G-protein Gq. We show that during long continued agonist action, polyphosphorylated inositol lipids are initially depleted but then regenerate while agonist is still present. Experiments and kinetic modeling reveal that the regeneration results from gradual but massive up-regulation of PI 4-kinase pathways rather than from desensitization of receptors. Golgi pools of phosphatidylinositol 4-phosphate and the lipid kinase PI4KIIIα (PI4KA) contribute to this homeostatic regeneration. This powerful acceleration, which may be at the level of enzyme activity or of precursor and product delivery, reveals strong regulatory controls in the phosphoinositide cycle.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa , Membrana Celular/química , Fosfatidilinositol 4,5-Difosfato , Fosfolipasas de Tipo CRESUMEN
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 Ca(2+) dynamics and insulin secretion in pancreatic ß cells.