Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld. Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld. Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld. Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld. This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.

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)P2degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P2depletion 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)P2sensitivity 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.

Voltage-gated Ca2+ channels contain β subunits that regulate channel gating. Park et al. conduct a comprehensive analysis of the role of the β subunit HOOK region and show that its B domain is important for PIP2 regulation of channel gating and that its A domain modulates this effect.

β subunits of high voltage-gated Ca 2+ (Ca V ) channels promote cellsurface 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 Ca V α1B-β interactions by rapamycin-translocatable Ca V β subunits that allow drug-induced sequestration and uncoupling of the β subunit from Ca V 2.2 channel complexes in intact cells. Without Ca V α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing Ca V α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B-β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from Ca V α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P 2 sensitivity. Furthermore, the mutations slow Ca V 2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in Ca V 2.3 channels. In sum, the strong interaction of Ca V α1B-β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells.voltage-gated Ca 2+ channel | Ca V β subunits | chemically inducible dimerization | rapamycin | PI(4,5)P 2 V oltage-gated Ca 2+ (Ca V ) channels play essential roles converting electrical signals to changes in Ca 2+ -dependent processes like synaptic transmission, muscle contraction, and gene transcription (1). The Ca V channels can be divided into high voltageactivated (HVA) (Ca V 1 and Ca V 2) and low voltage-activated (LVA) (Ca V 3) channels in accordance with their activation threshold. HVA Ca 2+ channels are composed of a pore-forming α1 subunit and at least three auxiliary subunits, the disulfide-linked complex of α2 and δ plus β. The auxiliary β subunit regulates cell surface trafficking and biophysical gating properties of HVA Ca 2+ channels via an interaction with the Ca V α1 subunit in 1:1 stoichiometry (2-4). Four distinct genes encode β1-β4 subunits and their splice variants (5-7). The β subunits contain a highly variable N and C terminus and a HOOK domain separating highly conserved src homology-3 (SH3) and guanylate kinase (GK) domains. The GK domain contains the α-binding pocket (ABP) that interacts directly with the α-interaction domain (AID) of the cytosolic I-II loop of Ca V α1 subunits (8-11). An addition...

Recently, we showed that the HOOK region of the b2 subunit electrostatically interacts with the plasma membrane and regulates the current inactivation and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) sensitivity of voltage-gated Ca 2C (Ca V ) 2.2 channels. Here, we report that voltage-dependent gating and current density of the Ca V 2.2 channels are also regulated by the HOOK region of the b2 subunit. The HOOK region can be divided into 3 domains: S (polyserine), A (polyacidic), and B (polybasic). We found that the A domain shifted the voltage-dependent inactivation and activation of Ca V 2.2 channels to more hyperpolarized and depolarized voltages, respectively, whereas the B domain evoked these responses in the opposite directions. In addition, the A domain decreased the current density of the Ca V 2.2 channels, while the B domain increased it.Together, our data demonstrate that the flexible HOOK region of the b2 subunit plays an important role in determining the overall Ca V channel gating properties.

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High-voltage-activated Ca2+ (CaV) channels that adjust Ca2+ influx upon membrane depolarization are differentially regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in an auxiliary CaV β subunit-dependent manner. However, the molecular mechanism by which the β subunits control the PIP2 sensitivity of CaV channels remains unclear. By engineering various α1B and β constructs in tsA-201 cells, we reported that at least two PIP2-binding sites, including the polybasic residues at the C-terminal end of I–II loop and the binding pocket in S4II domain, exist in the CaV2.2 channels. Moreover, they were distinctly engaged in the regulation of channel gating depending on the coupled CaV β2 subunits. The membrane-anchored β subunit abolished the PIP2 interaction of the phospholipid-binding site in the I–II loop, leading to lower PIP2 sensitivity of CaV2.2 channels. By contrast, PIP2 interacted with the basic residues in the S4II domain of CaV2.2 channels regardless of β2 isotype. Our data demonstrated that the anchoring properties of CaV β2 subunits to the plasma membrane determine the biophysical states of CaV2.2 channels by regulating PIP2 coupling to the nonspecific phospholipid-binding site in the I–II loop.

In this paper, we propose that it is necessary to expand the programs in the future, Iand it should be possible to activate and to participate the programs continuously, because the higher the participation in leisure program is, the higher life satisfaction.. The Korean Senior Citizens Association has a branch in each province and is used as a place where voluntarily provide friendship activities, hobbies for elderly people aged 65 and information exchange with other leisure activities. However, it was considered to be only a cursory space a few years ago, unlike the activation of leisure programs for the elderly welfare center. The purpose of this study is to investigate the effect of satisfaction of leisure program by type on life satisfaction of users who have experience in elderly university. The subjective health status and the residential type of participating elderly people differ in their life satisfaction. Therefore, it is important for the elderly to have regular health checkups and to maintain a stable residential style. And also, the experiment results show that life satisfaction was higher when leisure program use satisfaction was higher -especially, satisfaction with health program and health care program was higher. •Received: . 11. 23, Revised: 2016. 12. 05, Accepted: 2016 •This paper was supported(in part) by Research Funds of Kwangju Women's University in KWU116-015