| My research focuses on understanding the mechanisms of eukaryotic inwardly rectifying potassium channels (Kir), which are responsible for creating membrane potential stability. N- and C-terminal cytoplasmic domains of inwardly rectifying K (Kir) channels control the ion-permeation pathway through diverse interactions with small molecules and protein ligands in the cytoplasm. Two crystal structures of the cytoplasmic domains of Kir2.1 (Kir2.1L) and the G protein-sensitive Kir3.1 (Kir3.1S) channels in the absence of PIP2 show the cytoplasmic ion-permeation pathways occluded by four cytoplasmic loops that form a girdle around the central pore (G-loop). Significant flexibility of the pore-facing G-loop of Kir2.1L and Kir3.1S suggest a possible role as a diffusion barrier between cytoplasmic and transmembrane pores. Consistent with this, mutations of the G-loop disrupted gating or inward rectification. Structure comparison reveals a di-aspartate cluster on the distal end of the cytoplasmic pore of Kir2.1L that is important for modulating inward rectification. The Kir2.1L structural allows us the first structural look at the potential causes of Andersen's Syndrome, which is caused by defects in the inwardly rectifying potassium channel 2.1 (Kir2.1). We have characterized individual Andersen's Syndrome mutants R218Q, G300V, E303K, and Delta314-315 and have shown that they have multiple affects on the ability of Kir2.1 channel's cytoplasmic domains to assemble into proper tetrameric assemblies. The 2.0 A x-ray crystal structure of the N- and C-terminal cytoplasmic domains of Kir2.1 R218Q combined with T309K, reveals the G-loop and CD loop disruptions by the R218Q mutation. We show that E303 plays an important role in gating. Moreover, we demonstrate a novel DE loop stabilizing 2-methyl-2,4-pentanediol binding site, and cytoplasmic bound potassium ion, the first identified for this class of K channels. We have also conducted NMR studies on the last 58 residues of Kir2.1 to show the flexibility of this region and binding footprint of PSD95 PDZ domains, which tethers the channels to the cell's cytoskeleton. Taken together, these results demonstrate that cytoplasmic domains of Kir channels are structurally significant components of the channel and undergo structural changes to modulate gating and inward rectification. |
