Günter Schlichthörl

Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome.

Andersens syndrome, an autosomal dominant disorder related to mutations of the potassium channel Kir2.1, is characterized by cardiac arrhythmias, periodic paralysis, and dysmorphic bone structure. The aim of our study was to find out whether heteromerization of Kir2.1 channels with wild-type Kir2.2 and Kir2.3 channels contributes to the phenotype of Andersens syndrome. The following results show that Kir2.x channels can form functional heteromers: (i) HEK293 cells transfected with Kir2.x–Kir2.y concatemers expressed inwardly rectifying K+ channels with a conductance of 28–30 pS. (ii) Expression of Kir2.x–Kir2.y concatemers in Xenopus oocytes produced inwardly rectifying, Ba2+ sensitive currents. (iii) When Kir2.1 and Kir2.2 channels were coexpressed in Xenopus oocytes the IC50 for Ba2+ block of the inward rectifier current differed substantially from the value expected for independent expression of homomeric channels. (iv) Coexpression of nonfunctional Kir2.x constructs, in which the GYG region of the pore region was replaced by AAA, with wild-type Kir2.x channels produced both homomeric and heteromeric dominant-negative effects. (v) Kir2.1 and Kir2.3 channels could be coimmunoprecipitated in membrane extracts from isolated guinea pig cardiomyocytes. (vi) Yeast two-hybrid analysis showed interaction between the N- and C-terminal intracellular domains of different Kir2.x subunits. Coexpression of Kir2.1 mutants related to Andersens syndrome with wild-type Kir2.x channels showed a dominant negative effect, the extent of which varied between different mutants. Our results suggest that differential tetramerization of the mutant allele of Kir2.1 with wild-type Kir2.1, Kir2.2, and Kir2.3 channels represents the molecular basis of the extraordinary pleiotropy of Andersens syndrome.

Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea-pig cardiomyocytes.

1 The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea‐pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea‐pig cardiac ventricle. 2 A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea‐pig genomic library. 3 The cell‐specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi‐cell RT‐PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. 4 Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. 5 After transfection in human embryonic kidney cells (HEK293) the mean single‐channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. 6 Cell‐attached measurements in isolated guinea‐pig cardiomyocytes (n= 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. 7 Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half‐maximum block at ‐100 mV were 3.24 μm for gpKir2.1, 0.51 μm for gpKir2.2, 10.26 μm for gpKir2.3 and 235 μm for gpKir2.4. 8 Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell‐attached recordings. The concentration and voltage dependence of Ba2+ block of the large‐conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. 9 Our results suggest that the large‐conductance inward rectifier channels found in guinea‐pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate‐conductance (23.8 pS) and low‐conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively.

Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3.

The two‐pore‐domain potassium channels TASK‐1, TASK‐3 and TASK‐5 possess a conserved C‐terminal motif of five amino acids. Truncation of the C‐terminus of TASK‐1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP‐tagged channel proteins. Two‐hybrid analysis showed that the C‐terminal domain of TASK‐1, TASK‐3 and TASK‐5, but not TASK‐4, interacts with isoforms of the adapter protein 14‐3‐3. A pentapeptide motif at the extreme C‐terminus of TASK‐1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14‐3‐3, whereas the last 40 amino acids of TASK‐1 were required for strong binding. Deletion of a single amino acid at the C‐terminal end of TASK‐1 or TASK‐3 abolished binding of 14‐3‐3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK‐1 mutants that failed to interact with 14‐3‐3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK‐1 S409A, which interacts with 14‐3‐3‐like wild‐type channels, displayed normal macroscopic currents. Co‐injection of 14‐3‐3ζ cRNA increased TASK‐1 current in Xenopus oocytes by about 70 %. After co‐transfection in HEK293 cells, TASK‐1 and 14‐3‐3ζ (but not TASK‐1ΔC5 and 14‐3‐3ζ) could be co‐immunoprecipitated. Furthermore, TASK‐1 and 14‐3‐3 could be co‐immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14‐3‐3 with TASK‐1 or TASK‐3 may promote the trafficking of the channels to the surface membrane.

Expression pattern and functional characteristics of two novel splice variants of the two-pore-domain potassium channel TREK-2.

Two novel alternatively spliced isoforms of the human two‐pore‐domain potassium channel TREK‐2 were isolated from cDNA libraries of human kidney and fetal brain. The cDNAs of 2438 base pairs (bp) (TREK‐2b) and 2559 bp (TREK‐2c) encode proteins of 508 amino acids each. RT‐PCR showed that TREK‐2b is strongly expressed in kidney (primarily in the proximal tubule) and pancreas, whereas TREK‐2c is abundantly expressed in brain. In situ hybridization revealed a very distinct expression pattern of TREK‐2c in rat brain which partially overlapped with that of TREK‐1. Expression of TREK‐2b and TREK‐2c in human embryonic kidney (HEK) 293 cells showed that their single‐channel characteristics were similar. The slope conductance at negative potentials was 163 ± 5 pS for TREK‐2b and 179 ± 17 pS for TREK‐2c. The mean open and closed times of TREK‐2b at −84 mV were 133 ± 16 and 109 ± 11 μs, respectively. Application of forskolin decreased the whole‐cell current carried by TREK‐2b and TREK‐2c. The sensitivity to forskolin was abolished by mutating a protein kinase A phosphorylation site at position 364 of TREK‐2c (construct S364A). Activation of protein kinase C (PKC) by application of phorbol‐12‐myristate‐13‐acetate (PMA) also reduced whole‐cell current. However, removal of the putative TREK‐2b‐specific PKC phosphorylation site (construct T7A) did not affect inhibition by PMA. Our results suggest that alternative splicing of TREK‐2 contributes to the diversity of two‐pore‐domain K+ channels.

The Retention Factor p11 Confers an Endoplasmic Reticulum-Localization Signal to the Potassium Channel TASK-1

The interaction of the adaptor protein p11, also denoted S100A10, with the C‐terminus of the two‐pore‐domain K+ channel TASK‐1 was studied using yeast two‐hybrid analysis, glutathione S‐transferase pulldown, and co‐immunoprecipitation. We found that p11 interacts with a 40 amino‐acid region in the proximal C‐terminus of the channel. In heterologous expression systems, deletion of the p11‐interacting domain enhanced surface expression of TASK‐1. Attachment of the p11‐interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C‐terminal end of p11. Imaging of EGFP‐tagged TASK‐1 channels in COS cells suggested that wild‐type TASK‐1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK‐1 to the surface membrane. Our results suggest that binding of p11 to TASK‐1 retards the surface expression of the channel, most likely by virtue of a di‐lysine retention signal at the C‐terminus of p11. Thus, the cytosolic protein p11 may represent a ‘retention factor’ that causes localization of the channel to the ER.

Intracellular traffic of the K+ channels TASK-1 and TASK-3: role of N- and C-terminal sorting signals and interaction with 14-3-3 proteins.

The two‐pore‐domain potassium channels TASK‐1 (KCNK3) and TASK‐3 (KCNK9) modulate the electrical activity of neurons and many other cell types. We expressed TASK‐1, TASK‐3 and related reporter constructs in Xenopus oocytes, mammalian cell lines and various yeast strains to study the mechanisms controlling their transport to the surface membrane and the role of 14‐3‐3 proteins. We measured potassium currents with the voltage‐clamp technique and fused N‐ and C‐terminal fragments of the channels to various reporter proteins to study changes in subcellular localisation and surface expression. Mutational analysis showed that binding of 14‐3‐3 proteins to the extreme C‐terminus of TASK‐1 and TASK‐3 masks a tri‐basic motif, KRR, which differs in several important aspects from canonical arginine‐based (RxR) or lysine‐based (KKxx) retention signals. Pulldown experiments with GST fusion proteins showed that the KRR motif in the C‐terminus of TASK‐3 channels was able to bind to COPI coatomer. Disabling the binding of 14‐3‐3, which exposes the KRR motif, caused localisation of the GFP‐tagged channel protein mainly to the Golgi complex. TASK‐1 and TASK‐3 also possess a di‐basic N‐terminal retention signal, KR, whose function was found to be independent of the binding of 14‐3‐3. Suppression of channel surface expression with dominant‐negative channel mutants revealed that interaction with 14‐3‐3 has no significant effect on the dimeric assembly of the channels. Our results give a comprehensive description of the mechanisms by which 14‐3‐3 proteins, together with N‐ and C‐terminal sorting signals, control the intracellular traffic of TASK‐1 and TASK‐3.

A di-acidic sequence motif enhances the surface expression of the potassium channel TASK-3.

We have characterized a sequence motif, EDE, in the proximal C‐terminus of the acid‐sensitive potassium channel TASK‐3. Human TASK‐3 channels were expressed in Xenopus oocytes, and the density of the channels at the surface membrane was studied with two complementary techniques: a luminometric surface expression assay of hemagglutinin epitope‐tagged TASK‐3 channels and voltage‐clamp measurements of the acid‐sensitive potassium current. Both approaches showed that mutation of the two glutamate residues of the EDE motif to alanine (ADA mutant) markedly reduced the transport of TASK‐3 channels to the cell surface. Mutation of the central aspartate of the EDE motif had no effect on surface expression. The functional role of the EDE motif was further characterized in chimaeric constructs consisting of truncated Kir2.1 channels to which the C‐terminus of TASK‐3 was attached. In these constructs, too, replacement of the EDE motif by ADA strongly reduced surface expression. Live‐cell imaging of enhanced green fluorescent protein‐tagged channels expressed in COS‐7 cells showed that 24 h after transfection wild‐type TASK‐3 was mainly localized to the cell surface whereas the ADA mutant was largely retained in the endoplasmic reticulum (ER). Mutation of a second di‐acidic motif in the C‐terminus of TASK‐3 (DAE) had no effect on surface expression. Coexpression of TASK‐3 with a GTP‐restricted mutant of the coat recruitment GTPase Sar1 (Sar1H79G) resulted in ER retention of the channel. Our data suggest that the di‐acidic motif, EDE, in human TASK‐3 is a major determinant of the rate of ER export and is required for efficient surface expression of the channel.

TASK-1 Channels May Modulate Action Potential Duration of Human Atrial Cardiomyocytes

Background/Aims: Atrial fibrillation is the most common arrhythmia in the elderly, and potassium channels with atrium-specific expression have been discussed as targets to treat atrial fibrillation. Our aim was to characterize TASK-1 channels in human heart and to functionally describe the role of the atrial whole cell current ITASK-1. Methods and Results: Using quantitative PCR, we show that TASK-1 is predominantly expressed in the atria, auricles and atrio-ventricular node of the human heart. Single channel recordings show the functional expression of TASK-1 in right human auricles. In addition, we describe for the first time the whole cell current carried by TASK-1 channels (ITASK-1) in human atrial tissue. We show that ITASK-1 contributes to the sustained outward current IKsus and that ITASK-1 is a major component of the background conductance in human atrial cardiomyocytes. Using patch clamp recordings and mathematical modeling of action potentials, we demonstrate that modulation of ITASK-1 can alter human atrial action potential duration. Conclusion: Due to the lack of ventricular expression and the ability to alter human atrial action potential duration, TASK-1 might be a drug target for the treatment of atrial fibrillation.

A semisynthetic fusicoccane stabilizes a protein-protein interaction and enhances the expression of K+ channels at the cell surface

Small-molecule stabilization of protein-protein interactions is an emerging field in chemical biology. We show how fusicoccanes, originally identified as fungal toxins acting on plants, promote the interaction of 14-3-3 proteins with the human potassium channel TASK-3 and present a semisynthetic fusicoccane derivative (FC-THF) that targets the 14-3-3 recognition motif (mode 3) in TASK-3. In the presence of FC-THF, the binding of 14-3-3 proteins to TASK-3 was increased 19-fold and protein crystallography provided the atomic details of the effects of FC-THF on this interaction. We also tested the functional effects of FC-THF on TASK channels heterologously expressed in Xenopus oocytes. Incubation with 10 μM FC-THF was found to promote the transport of TASK channels to the cell membrane, leading to a significantly higher density of channels at the surface membrane and increased potassium current.

RNA editing modulates the binding of drugs and highly unsaturated fatty acids to the open pore of Kv potassium channels

The time course of inactivation of voltage‐activated potassium (Kv) channels is an important determinant of the firing rate of neurons. In many Kv channels highly unsaturated lipids as arachidonic acid, docosahexaenoic acid and anandamide can induce fast inactivation. We found that these lipids interact with hydrophobic residues lining the inner cavity of the pore. We analysed the effects of these lipids on Kv1.1 current kinetics and their competition with intracellular tetraethylammonium and Kvβ subunits. Our data suggest that inactivation most likely represents occlusion of the permeation pathway, similar to drugs that produce ‘open‐channel block’. Open‐channel block by drugs and lipids was strongly reduced in Kv1.1 channels whose amino acid sequence was altered by RNA editing in the pore cavity, and in Kv1.x heteromeric channels containing edited Kv1.1 subunits. We show that differential editing of Kv1.1 channels in different regions of the brain can profoundly alter the pharmacology of Kv1.x channels. Our findings provide a mechanistic understanding of lipid‐induced inactivation and establish RNA editing as a mechanism to induce drug and lipid resistance in Kv channels.