Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism.
Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays with two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins.
Saxena
Sunil K
SK
Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, USA. ssaxena@stevens.edu
Horiuchi
Hisanori
H
Fukuda
Mitsunori
M
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D053503:Epithelial Sodium Channels; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction / Q000502:physiology; D015222:Sodium Channels / Q000378:metabolism*; D050857:Synaptotagmins / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
D053503:Epithelial Sodium Channels; D008565:Membrane Proteins; D015222:Sodium Channels; C507969:UNC13D protein, human; D000075745:rab27 GTP-Binding Proteins; D050857:Synaptotagmins; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins