An Andersen-Tawil Syndrome Mutation in Kir2.1 (V302M) Alters the G-loop Cytoplasmic K+ Conduction Pathway

Abstract

Loss-of-function mutations in the inward rectifier potassium channel, Kir2.1, cause Andersen-Tawil syndrome (ATS-1), an inherited disorder of periodic paralysis and ventricular arrhythmias. Here, we explore the mechanism by which a specific ATS-1 mutation (V302M) alters channel function. Val-302 is located in the G-loop, a structure that is believed to form a flexible barrier for potassium permeation at the apex of the cytoplasmic pore. Consistent with a role in stabilizing the G-loop in an open conformation, we found the V302M mutation specifically renders the channel unable to conduct potassium without altering subunit assembly or attenuating cell surface expression. As predicted by the position of the Val-302 side chain in the crystal structure, amino acid substitution analysis revealed that channel activity and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity are profoundly sensitive to alterations in the size, shape, and hydrophobicity of side chains at the Val-302 position. The observations establish that the Val-302 side chain is a critical determinant of potassium conduction through the G-loop. Based on our functional studies and the cytoplasmic domain crystal structure, we suggest that Val-302 may influence PIP2 gating indirectly by translating PIP2 binding to conformational changes in the G-loop pore.

WNK4 Diverts the Thiazide-sensitive NaCl Cotransporter to the Lysosome and Stimulates AP-3 Interaction

“…Quantitative Antibody Binding Cell Surface Luminometry-2ϫHA-NCC cell surface expression was quantified by chemiluminescence using methods similar to those described previously (15). HEK-293H cells were transiently transfected at 90% confluence on 6-well Biocoat plates (BD Biosciences).…”

“…48 h later, cells were fixed in 4% paraformaldehyde, permeabilized, and blocked as described previously (15). Cells were then incubated with 3F10 (1:200), Myc (1:200), and LAMP-2 (1:30) antibodies in 5% FBS for 1 h at room temperature, washed 3 times in PBS, and incubated with Alexa 488, 568, and 635-conjugated secondary antibodies (all at 1:100 concentration in 0.5% FBS) for 30 min.…”

A comprehensive guide to the ROMK potassium channel: form and function in health and disease

“…Importantly, a Bartter mutation, A 306 T, that disrupts potassium conduction (107) is located in the G loop. Diseasecausing mutations in other K ir channels also affect the G loop, underscoring the general importance of the structure in the inward rectifiers (86,105).…”

“…3), it has been proposed that ligand binding (i.e., PIP 2 ) or phosphorylation at nearby sites on the surface of the cytoplasmic domain is allosterically coupled to TM2 movement through conformational changes at the G loop (86,97,106). Disruption of the coupling mechanism has been proposed to explain disease-causing gating defects in K ir 2.1 and K ir 6.2, involving mutations in G-loop residues (86,105,112). Obviously, further studies are required to critically test these ideas in ROMK.…”

Hypertension resistance polymorphisms in ROMK (Kir1.1) alter channel function by different mechanisms

“…This vector also contains a polyadenylate sequence in the 3=-UTR (dA23dC30). M 1 receptors were expressed in Xenopus oocytes as previously described (14). Oocytes were coinjected with M 1 and channel cRNA at a 5:1 ratio.…”