Dataset Information

RAB-10-GTPase-mediated regulation of endosomal phosphatidylinositol-4,5-bisphosphate.

ABSTRACT: Caenorhabditis elegans RAB-10 and mammalian Rab10 are key regulators of endocytic recycling, especially in the basolateral recycling pathways of polarized epithelial cells. To understand better how RAB-10 contributes to recycling endosome function, we sought to identify RAB-10 effectors. One RAB-10-binding partner that we identified, CNT-1, is the only C. elegans homolog of the mammalian Arf6 GTPase-activating proteins ACAP1 and ACAP2. Arf6 is known to regulate endosome-to-plasma membrane transport, in part through activation of type I phophatidylinositol-4-phosphate 5 kinase. Here we show that CNT-1 binds to RAB-10 through its C-terminal ankyrin repeats and colocalizes with RAB-10 and ARF-6 on recycling endosomes in vivo. Furthermore, we find that RAB-10 is required for the recruitment of CNT-1 to endosomal membranes in the intestinal epithelium. Consistent with negative regulation of ARF-6 by RAB-10 and CNT-1, we found overaccumulation of endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] in cnt-1 and rab-10 mutants and reduced endosomal PI(4,5)P2 levels in arf-6 mutants. These mutants produced similar effects on endosomal recruitment of the PI(4,5)P2-dependent membrane-bending proteins RME-1/Ehd and SDPN-1/Syndapin/Pacsin and resulted in endosomal trapping of specific recycling cargo. Our studies identify a RAB-10-to-ARF-6 regulatory loop required to regulate endosomal PI(4,5)P2, a key phosphoinositide in membrane traffic.

SUBMITTER: Shi A

PROVIDER: S-EPMC3435156 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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RAB-10-GTPase-mediated regulation of endosomal phosphatidylinositol-4,5-bisphosphate.

Shi Anbing A Liu Ou O Koenig Sabine S Banerjee Riju R Chen Carlos Chih-Hsiung CC Eimer Stefan S Grant Barth D BD

Proceedings of the National Academy of Sciences of the United States of America 20120806 35

Caenorhabditis elegans RAB-10 and mammalian Rab10 are key regulators of endocytic recycling, especially in the basolateral recycling pathways of polarized epithelial cells. To understand better how RAB-10 contributes to recycling endosome function, we sought to identify RAB-10 effectors. One RAB-10-binding partner that we identified, CNT-1, is the only C. elegans homolog of the mammalian Arf6 GTPase-activating proteins ACAP1 and ACAP2. Arf6 is known to regulate endosome-to-plasma membrane transp ...[more]

PMID: 22869721

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Project description:Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Shaker K+ channels and voltage-gated Ca2+ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP2 regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP2 regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K+ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP2 regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K+ channel family. Open-state stabilization by PIP2 has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP2 strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP2 has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4-S5 linker) and R479 near the S6 activation gate are required for PIP2 to inhibit voltage activation. The ability of PIP2 to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP2 on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP2-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP2 can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP2 regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.

Project description:Regulation of the putative amiloride and benzamil (Bz)-insensitive TRPV1t salt taste receptor by phosphatidylinositol 4,5-bisphosphate (PIP(2)) was studied by monitoring chorda tympani (CT) taste nerve responses to 0.1 M NaCl solutions containing Bz (5 x 10(-6) M; a specific ENaC blocker) and resiniferatoxin (RTX; 0-10 x 10(-6) M; a specific TRPV1 agonist) in Sprague-Dawley rats and in wildtype (WT) and TRPV1 knockout (KO) mice. In rats and WT mice, RTX elicited a biphasic effect on the NaCl + Bz CT response, increasing the CT response between 0.25 x 10(-6) and 1 x 10(-6) M. At concentrations >1 x 10(-6) M, RTX inhibited the CT response. An increase in PIP(2) by topical lingual application of U73122 (a phospholipase C blocker) or diC8-PIP(2) (a short chain synthetic PIP(2)) inhibited the control NaCl + Bz CT response and decreased its sensitivity to RTX. A decrease in PIP(2) by topical lingual application of phenylarsine oxide (a phosphoinositide 4 kinase blocker) enhanced the control NaCl + Bz CT response, increased its sensitivity to RTX stimulation, and inhibited the desensitization of the CT response at RTX concentrations >1 x 10(-6) M. The ENaC-dependent NaCl CT responses were not altered by changes in PIP(2). An increase in PIP(2) enhanced CT responses to sweet (0.3 M sucrose) and bitter (0.01 M quinine) stimuli. RTX produced the same increase in the Bz-insensitive Na(+) response when present in salt solutions containing 0.1 M NaCl + Bz, 0.1 M monosodium glutamate + Bz, 0.1 M NaCl + Bz + 0.005 M SC45647, or 0.1 M NaCl + Bz + 0.01 M quinine. No effect of RTX was observed on CT responses in WT mice and rats in the presence of the TRPV1 blocker N-(3-methoxyphenyl)-4-chlorocinnamide (1 x 10(-6) M) or in TRPV1 KO mice. We conclude that PIP(2) is a common intracellular effector for sweet, bitter, umami, and TRPV1t-dependent salt taste, although in the last case, PIP(2) seems to directly regulate the taste receptor protein itself, i.e., the TRPV1 ion channel or its taste receptor variant, TRPV1t.

Dataset Information

RAB-10-GTPase-mediated regulation of endosomal phosphatidylinositol-4,5-bisphosphate.

Publications

RAB-10-GTPase-mediated regulation of endosomal phosphatidylinositol-4,5-bisphosphate.

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