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. 2010 Nov 4;6(11):e1001200. doi: 10.1371/journal.pgen.1001200

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Kumar et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Homology model of the UNC-104 Pleckstrin homology (PH) domain docked with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂). Left: The original lesion in unc-104(e1265), D1497N, is marked along with the three residues (KK1463/4, R1496) known to be important for binding lipids. Note that the D1497N is a surface residue that lies in the same PI(4,5)P₂ binding pocket as the other three residues. Right: Positions of the two residues that are altered in the intragenic suppressors unc-104(e1265tb107) (having the R1501Q lesion) and unc-104(e1265tb120) (having the M1540I lesion) along with the original lesion D1497N. Blue indicates basic residues; red indicates acidic residues and pink indicates mildly acidic residues. (B) Percentage binding of the UNC-104 PH domain variants to various lipids in vitro. All data represented as mean ± SD and obtained from four independent experiments assayed in triplicates. (C) Normalized binding of PI(4,5)P₂ and brain lipids. Both D1497N and D1497N M1540I W1549A have very little specific binding to PI(4,5)P₂ or brain lipids when normalized to PC binding. However the D1497N R1501Q and D1497N M1540I variants bind significantly better than D1497N to PI(4,5)P₂. Data represented as mean ± SD (*p<0.005). PC – Phosphatidylcholine, PI(4,5)P₂ – Phosphatidyl inositol-4,5-bisphosphate. (D, E) Responsiveness of unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) to increase in PI(4,5)P₂ levels in vivo in all neurons. gqIs25 over-expresses Type I PIP kinase (ppk-1) phosphatidylinositol-4-phosphate 5′ kinase in neurons and increases PI(4,5)P₂ levels by 40% in vivo. (D) Locomotion of the various C. elegans strains. Wild type and gqIs25 animals move well. Worm locomotion is unchanged in unc-104(e1265) upon increase in PI(4,5)P₂. Locomotion is significantly improved in unc-104(e1265tb107) and unc-104(e1265tb120) when PI(4,5)P₂ levels are increased (*p<10^-5). n = 30 animals in all experiments. All data represented as mean ± SEM. (E) Aldicarb resistance of the various C. elegans strains as a measure of cholinergic transmission. Aldicarb inhibits acetylcholine esterase in C. elegans and causes hyperstimulation of the muscle and thus paralysis. Aldicarb resistance is unchanged in unc-104(e1265) upon increase in PI(4,5)P₂. Time for paralysis is reduced in unc-104(e1265tb107) and unc-104(e1265tb120) when PI(4,5)P₂ levels are increased (*p<0.001). n = 30 animals, done three times independently. Data represented as the average time (mean ± SEM) taken for 50% of the animals to be completely paralyzed. The alleles unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) are labeled in the figure by the respective protein changes they encode, namely D1497N, D1497N R1501Q and D1497N M1540I.