PIP2 Revision Flashcards
Phosphoinositides are
acidic phospholipids of the cytoplasmic leaflet of the membrane with myo-inositol head group
Parent compound of phosphoinositides
phosphatidylinositol - 3,4-5 - rise to 7 low abundance POLY phosphoinositides
Phosphoinositides 3 functionsq
- Mark identity of specific subcellular membrane components
- Membrane recognition sites for cytoplasmic proteins
- Membrane-delimited second messengers
PIP2 is
minority inner leaflet - regulates many ion channel + transporters whose activity is turned off by pathways that deplete PIP2
Who listed 2 functional advatnages
Suh and Hille 2008
First funcitonal advantage
Higgleman 1996
Silencing during transportatin and recycling
Second advantage
NT - increase PLC - regulated by incoming signals
PIP2 regulates which ion channels
- VGCC - N and P/Q
- TRP
- Two-P K
- Kir
- Transporters: NXC + PMCA
- Ca2+ release channels IP3 R + RYR
- Renal epithelial Na+ channels ENAC
Kir mutation
Plaster 2001 - exacerbated by enhanced sensitivity to PIP2 depletion by modulating stimuli
PIP2 does what to Kir’s
Stabilize Po
What residues in Kir’s are important for PIP2 binding?
- C-terminal fusion proteins - Huang 1998 bind directly (1.1, 2.1, 3.1,3.2)
- Homologous arginine in M2 of Kir 1.1, 2.1, 5.1,6.2 Huang 1998
- Lopes 2002 basic + hydrophobic residues in NTD and distal CTD
Implications of residues in PIP2 binding to Kir
Affinity + specificity to Kir2.1
Kir - apparent PIP2 affinity regulated by other signals - what are these?
- PKA - Kir 1.1 - Liou 1999 antibodies
- Kir 3.1-3.4 BY+ intracellular Na+ Huang, 1998
- Intracellular ATP Kir6.X - decrease PO Baukauwitz, 1998
Initial leads came from
- KirBac1.1 - inhibiited by PIP2 + 3 -> Kuo 2003 crystal structure
- CTD Kir 3.1/2.1 Nishida + MacKinnon 2002
First homology model
Logotethis 2007 - binding site is clustered in the same region in channel space as by and Na+ - regulates open.close and coordinates action of gating modifiers
Kir6.1
Analogous homology model
Haider - dock inositol phosphates surrogates for phosphoinositide head
Kir6.2
Final chimera
Membrane KirBac1.1. CTD Kir3.1
Ring-like cloud of basic residues near putative bilayer - likely binds
Homology model binding Kir6.2
4 binding sties each at interface between homotetrameric subunits - binds by contact with 6 basic residues and some H bonding
Maximal distance from bilayer - strain assists opening
Of all the ion channels regulated by PIP2,
PIP2 regulation of Kv7 most tied to physiological function
What type of GPCR inhbiits Kv7?
Delmas and Brown
Gq/11 suppresses M current by activation of PLC which depletes PIP2
What studies showed that time course of Kv7 inhibition coincides with PIP2 depletion?
- Horowitz 2005 - Gq/11 suppression
- Suh 2006 - inducible translocation of PIP2-5 phosphatase
10s
comparable to time taken for PIP2 depletion by these maneuvers
What showed that recovery of M-current was related to PIP2 resynthesis?
Suh and Hille, 2002 - 100-200 ms - requires PI4K and PI5K sequential action
What does direct application of PIP2 do?
Li 2005
increases pO and slows rundown
Why might PIP2 be the only phosphoinositide that acts on Kv7 in in tact cells?
- PIP2 - deplete engineered chemical dimerization system - suppress M - whereas augmented by PIP5 kinase Suh 2006
- PIP + PIP3 still complete inhibition in rapamycin system when these are activated by PIP2 - 5 phosphatase and 3 kinase
- PI 3,4 P2 - little affect on M current in excised patches
What do we know about the binding site for PIP2 on Kv7?
- polybasic residues in C terminus close to S6
- H328C point moutation - increase susceptibility to BKR mediated inhibition Zhang 2003
- candidate basic residues around this histidine
Overlap of binding site of PIP2 on Kv7?
- Kwon 2007 - 2 putative calmodulin binding sites- theme reported for other channels
- McLaughlin - many PIP2 binding proteins
- Gamper and Shapiro - 2003 -> calmodulin binding might modulate PIP2 binding
How is PIP3 made?
3’ phosphorylation by PI3K
What does PIP3 do?
PH and PH-like proteins to membrane surface (Englemann, 2006)
How does PH recruitment to membrane surface lead to Akt phosphorylation?
Proximity ->
- PI binding proteins - PDK-1 Alessi 1997
- Complexes such as mTorc2 - Sabrassov 2004/5
- > phosphorylate Akt
Effects of Akt
- Mostly inhibits
- Cell growth
- Cell cycle
- Cell survival
Example of Akt signalling
FKHR-L1
14-3-3 Brunet,2001
Retention in cytosol = no transcription of FKHR-L1 regulated genes
Intracellular calcium signals affect PIP2 how
- Depletion by Ga from Gq-GPCRi, Gby from GiS or Rac for PLCb2
- Accelerates PI kinases that lead to resynthesis Loewe 2007
Example of pathway that accelerates kinases
NCS-1 - wide sprectrum of actions - Burgogne + Weiss, 2001
Up regulation of PI4K - facilitate first step of PIP2 resynthesis Zhao,2001
PLC activated by
Ga - from Gq-GPCR
PLC does what?
binds PIP2 + translocates to surface membrane
at surface phase - 1000x fold greater concentration of PIP2 than in cytosol
depends on Ca2+ i activation
IP3 + DAG
DAG does what?
binds to C1 domain of PKC
PKC is
1/10 S/K - classified into 3 families based on ligand cofactor requirements - mediates Ca2+ downstream signalling
Consequences of PLC activation
- PIP2 depletion
- Calcium release
- PKC activation
- Channel inhibition
How do IP3R and RYR mediate differential responses?
- IP3 high freq. Ca2+ oscillations
2. RYR low freq - PKC -> CaMKII -> RYR
