Hum Mol Genet
September 15, 2014;
Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism-epilepsy phenotype.
Short QT3 syndrome (SQT3S) is a cardiac disorder characterized by a high risk of mortality and associated with mutations in Kir2.1
) channels. The molecular mechanisms leading to channel dysfunction, cardiac rhythm disturbances and neurodevelopmental disorders, potentially associated with SQT3S, remain incompletely understood. Here, we report on monozygotic twins displaying a short QT interval on electrocardiogram recordings and autism-epilepsy phenotype. Genetic screening identified a novel KCNJ2
variant in Kir2.1
that (i) enhanced the channel''s surface expression and stability at the plasma membrane
, (ii) reduced protein ubiquitylation and degradation, (iii) altered protein compartmentalization in lipid rafts by targeting more channels to cholesterol-poor domains and (iv) reduced interactions with caveolin 2. Importantly, our study reveals novel physiological mechanisms concerning wild-type Kir2.1
channel processing by the cell, such as binding to both caveolin 1 and 2, protein degradation through the ubiquitin-proteasome pathway; in addition, it uncovers a potential multifunctional site that controls Kir2.1
surface expression, protein half-life and partitioning to lipid rafts. The reported mechanisms emerge as crucial also for proper astrocyte
function, suggesting the need for a neuropsychiatric evaluation in patients with SQT3S and offering new opportunities for disease management.
Hum Mol Genet
ATP-sensitive potassium channel complex
Disease Ontology terms:
short QT syndrome
SHORT QT SYNDROME 3; SQT3
[+] show captions
Figure 1. Mutation detection by sequence analysis of the KCNJ2 coding region. (A) Pedigree of the family harboring a novel mutations in KCNJ2. Squares are males and circles females; solid black symbols represent propositi and slash deceased individual. (B) ECG recording from propositi showing shortened QT interval (heart rate, 87 bpm; QT 275 ms; paper speed 25 mm/s). (C) Electropherograms showing the heterozygous c.1037A>C transition (arrow-headed), predicting a novel non-synonymous p.K346T variant in propositi compared with the sequence of a healthy individual (WT). (D) Alignments of several KCNJ2 sequences flanking the K346T substitution (K/T, arrow-headed) showing that this residue is highly conserved in several vertebrate species. (E) Schematic representation of the membrane topology of a human Kir2.1 subunit indicating the position of the p.K346T variant.
Figure 2. The K346T mutation increases Kir2.1 currents. Sample current families recorded from oocytes expressing WT (A) or K346T (B) mRNA (the pulse protocol is shown as inset). (C) I–V relationships for WT (white circles, 3 ng) and K346T (black circles, 3 ng) channels. (D) Current amplitudes recorded at −100 mV from cells injected with the indicated mRNAs whose amounts are reported in brackets (mean ± SEM; n = 120; *P < 0.05; **P < 0.01). (E) Current amplitudes recorded at −60 mV for WT (white circles) and K346T (black circles) channels and plotted as a function of time after mRNA injection (mean ± SEM; n = 6).
Figure 3. Characterization of astrocytoma cells expressing WT and K346T channels. Co-immunofluorescences of cells expressing WT (A) or K346T (B) channels with anti-Kir2.1 pAb (red) and FITC-conjugated phallacidin (green) show that WT channels are localized in perinuclear vesicles (short arrows in A) and occasionally at plasma membranes (long arrows in A), while mutated channels are mainly expressed at plasma membranes (long arrows in B). Scale bar: 10 μm. (C) RT-PCR analysis of Kir2.1 mRNA in WT (1), K346T (2) channel or empty-vector expressing U251 cell lines (3). GAPDH housekeeping gene normalizes the amount of template. (D) WB analysis of membrane (MEM) and cytosolic (CYT) proteins derived from WT or K346T Kir2.1-expressing cells after Histidine co-purification. Molecular weight markers are on the left (kDa). (E) Densitometric analysis of protein bands from four independent experiments (mean ± SEM, *P < 0.05). (F) The resting membrane potential and (G) current density (at −100 mV) were evaluated in cells expressing WT (white bars) or K346T (gray bars) channels (data are mean ± SEM; n = 6; *P < 0.05; **P < 0.01).
Figure 4. The K346T mutation increases protein stability. (A) WB analysis of protein extracts derived from cells expressing WT and K346T channels treated with the protein synthesis inhibitor cycloheximide for 3, 6 and 12 h. WT protein degradation is almost complete after 12 h treatment, while K346T protein is still detectable at this time. Actin is used as loading control. Molecular weight markers are on the left (kDa). (B) Densitometric analysis of protein bands normalized with respect to the amount of either WT (white bar) or K346T (gray bar) Kir2.1 protein in control conditions. Data are expressed as mean ± SEM from four independent experiments (***P < 0.001).
Figure 5. The K346T mutation affects the distribution of Kir2.1 channels in membrane lipid rafts. (A) WB analysis of cholesterol-rich (triton insoluble fractions: 3–5) and cholesterol-poor membrane fractions (triton soluble fractions: 10–12) of WT or K346T Kir2.1-expressing cells. WT channels are mainly distributed in triton insoluble fractions (gray box), whereas K346T is also abundantly localized in cholesterol-poor fractions (black boxes). Cav-1 and flotillin-1 identify the caveolar raft fractions. Molecular weight markers are on the left (kDa). (B–E) Normal distributions of total protein (indicated on top) in membrane fractions isolated by sucrose density gradient. The levels of protein in each fraction are normalized to the total protein amount recovered from all the fractions together.
Figure 6. The K346T mutation reduces Kir2.1 channels interaction with Cav-2. (A) WB analysis of Kir2.1 channel's interactors after Histidine (His) co-purification of astrocytoma cells expressing WT or K346T channels. Input lanes represent protein extracts before His co-purification. WT and K346T channels co-purify similarly with Cav-1, whereas the K346T mutation reduces the association with Cav-2. One representative experiment out of three is shown. Molecular weight markers are indicated on the left (kDa). (B) Densitometric analysis (ratio) of protein bands corresponding to Cav-1 and Cav-2 normalized respect to WT (white bars) or K346T (gray bars) Kir2.1 protein levels. Data are expressed as mean ± SEM from three independent experiments (***P < 0.001).