PIP2 Revision

Question 1

Phosphoinositides are

Answer 1

acidic phospholipids of the cytoplasmic leaflet of the membrane with myo-inositol head group

Question 2

Parent compound of phosphoinositides

Answer 2

phosphatidylinositol - 3,4-5 - rise to 7 low abundance POLY phosphoinositides

Question 3

Phosphoinositides 3 functionsq

Answer 3

1. Mark identity of specific subcellular membrane components
2. Membrane recognition sites for cytoplasmic proteins
3. Membrane-delimited second messengers

Question 4

PIP2 is

Answer 4

minority inner leaflet - regulates many ion channel + transporters whose activity is turned off by pathways that deplete PIP2

Question 5

Who listed 2 functional advatnages

Answer 5

Suh and Hille 2008

Question 6

First funcitonal advantage

Answer 6

Higgleman 1996

Silencing during transportatin and recycling

Question 7

Second advantage

Answer 7

NT - increase PLC - regulated by incoming signals

Question 8

PIP2 regulates which ion channels

Answer 8

1. VGCC - N and P/Q
2. TRP
3. Two-P K
4. Kir
5. Transporters: NXC + PMCA
6. Ca2+ release channels IP3 R + RYR
7. Renal epithelial Na+ channels ENAC

Question 9

Kir mutation

Answer 9

Plaster 2001 - exacerbated by enhanced sensitivity to PIP2 depletion by modulating stimuli

Question 10

PIP2 does what to Kir’s

Answer 10

Stabilize Po

Question 11

What residues in Kir’s are important for PIP2 binding?

Answer 11

1. C-terminal fusion proteins - Huang 1998 bind directly (1.1, 2.1, 3.1,3.2)
2. Homologous arginine in M2 of Kir 1.1, 2.1, 5.1,6.2 Huang 1998
3. Lopes 2002 basic + hydrophobic residues in NTD and distal CTD

Question 12

Implications of residues in PIP2 binding to Kir

Answer 12

Affinity + specificity to Kir2.1

Question 13

Kir - apparent PIP2 affinity regulated by other signals - what are these?

Answer 13

1. PKA - Kir 1.1 - Liou 1999 antibodies
2. Kir 3.1-3.4 BY+ intracellular Na+ Huang, 1998
3. Intracellular ATP Kir6.X - decrease PO Baukauwitz, 1998

Question 14

Initial leads came from

Answer 14

1. KirBac1.1 - inhibiited by PIP2 + 3 -> Kuo 2003 crystal structure
2. CTD Kir 3.1/2.1 Nishida + MacKinnon 2002

Question 15

First homology model

Answer 15

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

Question 16

Analogous homology model

Answer 16

Haider - dock inositol phosphates surrogates for phosphoinositide head

Kir6.2

Question 17

Final chimera

Answer 17

Membrane KirBac1.1. CTD Kir3.1

Ring-like cloud of basic residues near putative bilayer - likely binds

Question 18

Homology model binding Kir6.2

Answer 18

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

Question 19

Of all the ion channels regulated by PIP2,

Answer 19

PIP2 regulation of Kv7 most tied to physiological function

Question 20

What type of GPCR inhbiits Kv7?

Answer 20

Delmas and Brown

Gq/11 suppresses M current by activation of PLC which depletes PIP2

Question 21

What studies showed that time course of Kv7 inhibition coincides with PIP2 depletion?

Answer 21

1. Horowitz 2005 - Gq/11 suppression
2. Suh 2006 - inducible translocation of PIP2-5 phosphatase
   10s
   comparable to time taken for PIP2 depletion by these maneuvers

Question 22

What showed that recovery of M-current was related to PIP2 resynthesis?

Answer 22

Suh and Hille, 2002 - 100-200 ms - requires PI4K and PI5K sequential action

Question 23

What does direct application of PIP2 do?

Answer 23

Li 2005

increases pO and slows rundown

Question 24

Why might PIP2 be the only phosphoinositide that acts on Kv7 in in tact cells?

Answer 24

1. PIP2 - deplete engineered chemical dimerization system - suppress M - whereas augmented by PIP5 kinase Suh 2006
2. PIP + PIP3 still complete inhibition in rapamycin system when these are activated by PIP2 - 5 phosphatase and 3 kinase
3. PI 3,4 P2 - little affect on M current in excised patches

Question 25

What do we know about the binding site for PIP2 on Kv7?

Answer 25

1. polybasic residues in C terminus close to S6 2. H328C point moutation - increase susceptibility to BKR mediated inhibition Zhang 2003 3. candidate basic residues around this histidine

Question 26

Overlap of binding site of PIP2 on Kv7?

Answer 26

1. Kwon 2007 - 2 putative calmodulin binding sites- theme reported for other channels 2. McLaughlin - many PIP2 binding proteins 3. Gamper and Shapiro - 2003 -> calmodulin binding might modulate PIP2 binding

Question 27

How is PIP3 made?

Answer 27

3' phosphorylation by PI3K

Question 28

What does PIP3 do?

Answer 28

PH and PH-like proteins to membrane surface (Englemann, 2006)

Question 29

How does PH recruitment to membrane surface lead to Akt phosphorylation?

Answer 29

Proximity -> 1. PI binding proteins - PDK-1 Alessi 1997 2. Complexes such as mTorc2 - Sabrassov 2004/5 - > phosphorylate Akt

Question 30

Effects of Akt

Answer 30

1. Mostly inhibits 2. Cell growth 3. Cell cycle 4. Cell survival

Question 31

Example of Akt signalling

Answer 31

FKHR-L1 14-3-3 Brunet,2001 Retention in cytosol = no transcription of FKHR-L1 regulated genes

Question 32

Intracellular calcium signals affect PIP2 how

Answer 32

1. Depletion by Ga from Gq-GPCRi, Gby from GiS or Rac for PLCb2 2. Accelerates PI kinases that lead to resynthesis Loewe 2007

Question 33

Example of pathway that accelerates kinases

Answer 33

NCS-1 - wide sprectrum of actions - Burgogne + Weiss, 2001 Up regulation of PI4K - facilitate first step of PIP2 resynthesis Zhao,2001

Question 34

PLC activated by

Answer 34

Ga - from Gq-GPCR

Question 35

PLC does what?

Answer 35

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

Question 36

DAG does what?

Answer 36

binds to C1 domain of PKC

Question 37

PKC is

Answer 37

1/10 S/K - classified into 3 families based on ligand cofactor requirements - mediates Ca2+ downstream signalling

Question 38

Consequences of PLC activation

Answer 38

1. PIP2 depletion 2. Calcium release 3. PKC activation 4. Channel inhibition

Question 39

How do IP3R and RYR mediate differential responses?

Answer 39

1. IP3 high freq. Ca2+ oscillations | 2. RYR low freq - PKC -> CaMKII -> RYR