Structural determinants and specificities for ROMK1-phosphoinositide interaction.
We have recently reported that direct interaction between phosphatidylinositol bisphosphate (PIP(2)) and the COOH-terminal cytoplasmic domain of ROMK1 is important for opening of the channel. We identified arginine-188 of ROMK1 as a critical residue for this interaction. Here, we further report that substitution of a neutral amino acid for lysine-181, arginine-217, or lysine-218 decreases single-channel open probability for the full-conductance state and increases the frequency of opening at a subconductance state. Compared with wild-type ROMK1 channels, these substitution mutants also display an increased sensitivity to the block by anti-PIP(2) antibodies and to inhibition by intracellular protons. These results indicate that, like arginine-188, lysine-181, arginine-217, and lysine-218 are also involved in interactions with PIP(2) and are critical for ROMK1 to open at full conductance. Using synthetic phosphoinositides containing phosphates at different positions in the head group, we also examined the specificities of phosphoinositides in the regulation of ROMK1 channels. We found that phosphoinositides containing phosphate at both positions 4 and 5 of the inositol head group have the highest efficacy in activating ROMK1 channels. These results suggest that phosphatidylinositol 4,5-bisphosphate is likely the important phosphoinositide in the regulation of ROMK1 channels in a physiological membrane milieu.
Zeng
Wei-Zhong
WZ
Department of Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8856, USA.
Liou
Horng-Huei
HH
Krishna
U Murali
UM
Falck
J R
JR
Huang
Chou-Long
CL
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001120:Arginine; D001665:Binding Sites; D002417:Cattle; D004553:Electric Conductivity; D004563:Electrochemistry; D005973:Glutamine; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000276:immunology / Q000378:metabolism; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D013329:Structure-Activity Relationship; D014162:Transfection; D014982:Xenopus laevis
D000906:Antibodies; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D005973:Glutamine; D001120:Arginine; D008239:Lysine; D000409:Alanine