Illuminating kir channel function in Anderson-Tawil syndrome.

Anderson–Tawil syndrome type 1 (ATS1) is a rare, autosomal dominant disorder, characterized by a variety of facial and skeletal dysmorphisms, periodic muscular weakness, and frequent ventricular arrhythmias. The disease is caused by loss-of-function (LOF) mutations in the KCNJ2 gene, which encodes for the pore-forming subunits of the inward rectifier potassium channel Kir2.1. Each Kir2.1 subunit consists of intracellular Nand C-terminal domains and two transmembrane helices linked by an extracellular Kþ-selective pore region (Figure 1A). Four subunits assemble to form phospholipid-gated channel tetramers that are critical for membrane potential control in skeletal and cardiac muscle as well as in ectodermal tissue that gives rise to craniofacial structures during development. ATS1-linked mutations cluster in two regions of Kir2.1 subunits: the selectivity filter pore loop and the binding sites for the activating ligand phosphatidylinositol-4,5-bisphosphate (PIP2) in the cytoplasmic domains (CD-I) (Figure 1A). Notably, interunit salt bridges at the interface of the CD-I of adjacent subunits (Figure 1B) have been shown to be important for normal Kir2.1 channel activity. The detailed molecular mechanisms by which Kir2.1 mutations associated with ATS1 give rise to channel dysfunction remain poorly understood. Writing in Cardiovascular Research, Déri and colleagues report that the novel ATS1-linked Kir2.1 LOF mutation p. Glu293Lys (E293K) reduces inward rectifier Kþ currents.Specifically, mimicking heterozygosity for the E293K mutation by co-transfecting wild-type and E293K Kir2.1 channels in human embryonic kidney (HEK) 293 cells resulted in whole-cell inward rectifier current densities that were significantly smaller compared to those in cells transfected with wild-type channels only, while transfection with mutant channels only did not result in measurable inward rectifier currents. These observations confirmed the LOF of the E293K mutation and its dominant negative effect over wild-type channels. Next, the authors examined whether surface expression deficits through reduced membrane trafficking contributes to the LOF phenotype. Confocal immunofluorescence microscopy revealed partial redistribution of Kir2.1 immunoreactivity to a submembrane space in cells transfected with E293K Kir2.1, compatible with reduced Kir anchoring in the plasmalemma. Finally, the authors used luminescence-based binary reporter assays to address the intriguing hypothesis that the mutation-induced charge reversal at the 293 position disrupts predicted salt bridges at the interface of the CD-I of adjacent subunits, reducing channel open probability. HEK 293 cells were transfected with cDNAs encoding for wild-type and/or mutant Kir2.1 subunits whose cytoplasmic C-termini are fused to functionally complementary small (Sm) or large (Lg) fragments of the catalytic domain of the enzyme luciferase (Figure 1C). Only complementary subunits that are within nanometre distance from one another can form catalytically active luciferase. Thus, the magnitude of luminescence intensity generated by complemented luciferase upon exposure to the substrate furimazine is thought to reflect connectivity between adjacent subunits within oligomers. Luminescence intensity in cells transfected with Smand Lg-tagged E293K Kir2.1 was found to be significantly lower compared to cells transfected with complementing wild-type Kir2.1 reporters, and indistinguishable from that in negative control cells (which co-expressed non-interacting Sm-tagged wt Kir2.1 and Lg-tagged KvLQT1 subunits) (Figure 1C). Co-expression of Lg-tagged E293K Kir2.1 with Sm-tagged wt Kir2.1 gave rise to intensity values that were intermediate between those in cells expressing homomeric wt Kir2.1 or heteromeric wt Kir2.1-KvLQT1 reporter pairs (Figure 1C). Luminescence intensity in cells transfected with ‘snorkel’ versions of heteromeric wt Kir2.1-E293K Kir2. reporter pairs was similar to that in cells transfected with homomeric ‘snorkel’ wt Kir2.1 reporter pairs (Figure 1D), confirming their anchoring in the surface membranes. Taken together, these results lead the authors to suggest a working model where charge reversal at E293 weakens electrostatic contacts at the CD-I of adjacent subunits, causing their relative dissociation from one another and reducing Kir2.1 channel activity. Alterations in Kir channel gating would then conspire with deficits in Kir membrane trafficking to give rise to the LOF phenotype of the E293K Kir2.1 mutation. How can disruption of CD-I salt bridges reduce Kir channel activity? Using targeted mutagenesis in conjunction with the patch-clamp technique, the Nichols laboratory recently demonstrated that neutralization of certain positively charged residues at the CD-I induces Kir channel