PIP2 Kir's

Question 1

Which channels were first recognized as PIP2 dependent (who)?

Answer 1

Kir6.x (KATP) channels were the first channels recognized as PIP2 dependent (Hilgemann, 1996)

Question 2

What kind of channels are Kir6.x’s ?

Answer 2

They are members of the larger superfamily of inward rectifier channels all of which may require PIP2 for function.

Question 3

How has Kir channel PIP2 activity been shown important in humans (who)?

Answer 3

Some mutant Kir channels associated with inherited diseases have increased susceptibility to modulation by stimuli that decrease membrane PIP2 levels, exacerbating the disease phenotype (Plaster, 2001).

Question 4

How do Kir channels depend on PIP2?

Answer 4

In general, Kir channels run down if PIP2 is depleted and reactivate if PIP2 micelles are applied.

Question 5

How does PIP2 react with Kir’s?

Answer 5

PIP2 stabilizes the open state. C-terminal fusion proteins made from Kir1.1 (ROMK1), Kir2.1 (IRK1), Kir3.1 (GIRK1), or Kir3.2 (GIRK2) channels bind PIP2 directly (Huang, 1998).

Question 6

What mutagenesis study has shown us the residue underlying PIP2 affinity in Kir’s?

Answer 6

Mutating a homologous arginine in the second transmembrane segment of Kir1.1, Kir2.1, Kir5.1, and Kir6.2 reduces the apparent affinity for PIP2 (Huang, 1998).

Question 7

Is arginine the only reside implicated in PIP2 binding?

Answer 7

Other basic and hydrophobic residues in the N terminus and distal C terminus are implicated in PIP2 binding to the channels (Lopes, 2002)

Question 8

What is the significance of variations in PIP2 binding residues in Kir?

Answer 8

They may enhance the specificity and affinity toward PIP2 in Kir2.1 compared to other types of Kir channels.

Question 9

Besides amino acid composition, what influences PIP2 binding to Kir?

Answer 9

Other signals regulate apparent affinity

Question 10

What signals regulate Kir1.1 affinity for PIP2?

Answer 10

Phosphorylation of Kir1.1 channels by protein kinase A (PKA) increases the apparent affinity for PIP2, making the current less sensitive to inhibition by PIP2 antibody (Liou, 1999)

Question 11

What signals regulate Kir3.1/4 affinity for PIP2?

Answer 11

Affinity of Kir3.1/4 (GIRK1/4) channels for PIP2 is increased by the Gβγ subunits of GTP-binding proteins and by intracellular Na+ (Huang, 1998)

Question 12

What signals regulate Kir6.x affinity for PIP2?

Answer 12

PIP2 interaction with Kir6.x channels is reduced by intracellular ATP, reducing the probability of opening (Baukrowitz, 1998)

Question 13

What were the initial leads to a 3D structure of PIP2 binding sites in Kir channels?

Answer 13

1. a crystal structure of a prokaryotic Kir channel KirBac1.1 that is inhibited by PIP2 and PIP3 (Kuo, 2003)
2. structures of cytoplasmic domains of Kir3.1 and Kir 2.1 (Nishida and MacKinnon, 2002)

Question 14

What did initial crystal structures lead to?

Answer 14

From these, Logothetis et al. (2007) developed a homology model for the Kir3.1 channel and concluded that residues implicated in PIP2 binding and residues implicated in the actions of several other modulatory agents (Na+, Gβγ) cluster in the same region of the channel in space

Question 15

What might the region containing residues for PIP2 binding + modulatory agents confer?

Answer 15

Presumably this region closely affects the balance between open and closed channels and coordinates the actions of many gating modifiers

Question 16

What homology model of what channel was made at the same time as Logothetis’s?

Answer 16

Analogously, Haider et al. (2007) made a homology model of Kir6.2

Question 17

What did Haider manage to do with his model?

Answer 17

successfully docked inositol phosphates as surrogates for phosphoinositide head groups with the model

Question 18

What finally determined the PIP2 binding site?

Answer 18

Nishida et al. (2007) obtained the crystal structure of a chimera of the membrane portion of KirBac1.1 and the cytoplasmic portion of Kir3.1

Question 19

What did Nishida’s crystal structure show (2007)?

Answer 19

a ring-like cloud of basic residues a small distance from the putative bilayer that might bind to phosphoinositides

Question 20

Which model precisely predicts where PIP2’s may bind and where does it suggest?

Answer 20

In the homology model for Kir6.1 there are four PIP2 binding sites, each at the interface between the homotetrameric subunits where gating conformational shifts might occur

Question 21

What would PIP2 be bound to? as suggested by the Kir6.1 homology model -

Answer 21

The phosphoinositide would be bound by contact with six basic residues and some hydrogen-bond interactions (Haider, 2007).

Question 22

Where would the PI head group be and what are the implications of this on Kir?

Answer 22

The phosphoinositide head group is near its maximum distance from the hydrophobic bilayer, which might imply that it pulls like a spring on the structure (near the slide helix) and this strain assists channel opening