Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-013-1404-z
ION CHANNELS, RECEPTORS AND TRANSPORTERS
Breaking the silence: functional expression of the two-pore-domain potassium channel THIK-2 Vijay Renigunta & Xinle Zou & Stefan Kling & Günter Schlichthörl & Jürgen Daut
Received: 8 November 2013 / Accepted: 9 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract THIK-2 belongs to the ‘silent’ channels of the twopore-domain potassium channel family. It is highly expressed in many nuclei of the brain but has so far resisted all attempts at functional expression. THIK-2 has a highly conserved 19-amino-acid region in its N terminus (residues 6–24 in the rat orthologue) that is missing in the closely related channel THIK-1. After deletion of this region (THIK-2Δ6–24 mutant), functional expression of the channel was observed in Xenopus oocytes and in mammalian cell lines. The resulting potassium current showed outward rectification under physiological conditions and slight inward rectification with symmetrical highK+ solutions and could be inhibited by application of halothane or quinidine. Another THIK-2 mutant, in which the putative retention/retrieval signal RRR at positions 14-16 was replaced by AAA, produced a similar potassium current. Both mutants showed a distinct localisation to the surface membrane when tagged with green fluorescent protein and expressed in a mammalian cell line, whereas wild-type THIK2 was mainly localised to the endoplasmic reticulum. These findings suggest that deletion of the retention/retrieval signal RRR enabled transport of THIK-2 channels to the surface membrane. Combining the mutation THIK-2Δ6–24 with a mutation in the pore cavity (rat THIK-2I158G) gave rise to a 12-fold increase in current amplitude, most likely due to an increase in open probability. In conclusion, the characteristics of THIK-2 channels can be analysed in heterologous expression systems by using trafficking and/or gating mutants. The possible mechanisms that enable THIK-2 expression at the surface membrane in vivo remain to be determined.
V. Renigunta (*) : X. Zou : S. Kling : G. Schlichthörl : J. Daut (*) Institute of Physiology and Pathophysiology, Marburg University, Deutschhausstr. 2, 35037 Marburg, Germany e-mail: [email protected] e-mail: [email protected]
Keywords K2P channels . Retention signal . Trafficking . Halothane
Introduction The family of two-pore-domain potassium channels (K2P channels) has fifteen members [7]. Five of these channels, TWIK-1, TWIK-2, KCNK7, TASK-5, and THIK-2, were originally regarded as ‘silent’ channels because no currents associated with these channel proteins could be recorded in native cells and no currents (or only very small currents) could be measured in heterologous expression systems. The reason for the silence of TWIK-1 has recently been detected: It carries an endocytosis signal (DxxxII) that prevents trafficking to the surface membrane. After mutation of the two isoleucine residues, the channel was transported to the surface membrane, and the properties of TWIK-1 currents could be analysed [8]. The currents carried by TWIK-2 [5], KCNK7 [16], and TASK-5 [11] channels are still elusive. In the present study, we tried to elucidate the characteristics of THIK-2 channels [14]. Like all other K2P channels, THIK-2 has two pore domains (P1 and P1) and a long M1–P1 linker. It has a long cytosolic C terminus and a very unusual N-terminal domain containing many proline, arginine, and cysteine residues [14]. We report here that in heterologous expression systems, only a very small fraction of THIK-2 channels is localised to the surface membrane and that THIK-2 channels are efficiently transported to the cell surface when a retention/retrieval signal (RRR) in its N terminus is mutated. Another mutation in the pore domain of THIK-2 appeared to cause an increase in the open probability of the channel. The trafficking mutant and the gating mutants allowed us to describe for the first time the characteristics of THIK-2 channels. Since THIK-2 is highly and differentially expressed in many regions of the brain
Pflugers Arch - Eur J Physiol
[12, 14] as well as in lung, liver, and kidney, it may be assumed that the potassium currents carried by THIK-2 channels are functionally important under some physiological or pathophysiological conditions.
Materials and methods Ethical approval For experiments involving Xenopus oocytes, adult female African clawed frogs (Xenopus laevis) were used. The frogs were anaesthetised by putting them in water containing 1 g/l tricaine. Stage V oocytes were obtained from ovarian lobes. Anaesthesia and operation were carried out in accordance with the principles of German legislation with approval of the animal welfare officer of the Medical Faculty of Marburg University under the governance of the Regierungspräsidium Giessen (the regional veterinary heath authority).
anti-rat immunoglobulin G antibody (Jackson ImmunoResearch) in 1 % BSA/ND96 solution for 60 min. Oocytes were washed thoroughly, initially in 1 % BSA/ND96 (at 4 °C for 60 min) and then in ND96 without BSA (at 4 °C for 15 min). Individual oocytes were placed in 20 μl SuperSignal Elisa Femto solution (Pierce Protein Biology Products, Rockford, IL, USA), and chemiluminescence was quantified in a luminometer (Lumat LB9507, Berthold Technologies). The luminescence produced by un-injected oocytes was used as a reference signal (negative control). To analyse the functionality of the endoplasmic reticulum (ER) localisation signal in the N terminus of THIK-2, we fused the N terminus of THIK-2 with the type 2 protein CD74. The surface expression of this reporter protein construct was determined in COS-7 cells using a luminometric assay described previously [24]. Based on previous experiments with CD74 [17], the N-terminal sequence MHRRRSR was used as a positive control (indicating retention), and CD74 with the mutated N-terminal sequence MHSSSSS was used as a negative control (indicating surface expression).
Molecular cloning and mutagenesis Cell culture Full-length rat THIK-1 (accession no. NM_022293) and rat THIK-2 (accession no. NM_022292) channels cloned into the pSGEM oocyte expression vector were used for voltageclamp experiments [14]. QuikChange (Agilent Technologies) was used to introduce deletions, insertions, and point mutations. For CD74 reporter assays, the N terminus of THIK-2 was fused to the reporter protein CD74. For luminometric measurements of surface expression in Xenopus oocytes, we used a pSGEM vector with THIK-1 or THIK-2 containing an extracellular haemagglutinin (HA) tag (THIK-1 at position 95 and THIK-2 at position 114). The surface membrane marker GFP-C1-PLCδ-PH (the pleckstrin homology domain of phospholipase C delta 1 [20]) was obtained from the non-profit clone repository AddGene (ID: 21179) and later subcloned to the pDsRed-Monomer-C1 vector for membrane co-localisation experiments. All DNA constructs were verified by sequencing. Surface expression assay The surface expression of HA-tagged THIK-1 and THIK-2 channels in Xenopus oocytes was analysed 2 days after injection of cRNA (6 ng/oocyte). Oocytes were incubated for 30 min in an ND96 solution containing 1 % bovine serum albumin (BSA) at 4 °C to block non-specific binding of antibodies. Subsequently, oocytes were incubated for 60 min at 4 °C with 100 μg/ml of rat monoclonal anti-HA antibody (clone 3F10, Roche) in 1 % BSA/ND96, washed six times at 4 °C with 1 % BSA/ND96, and incubated with 0.8 mg/ml peroxidase-conjugated affinity-purified F(ab)2 fragment goat
HeLa and COS-7 cells were cultured in high-glucose DMEM, 10 % foetal calf serum (FCS) (Life Technologies, Paisley, UK) and 1 % penicillin/streptomycin (PAA Laboratories GmbH, Pasching, Austria). Chinese hamster ovary (CHO) cells were cultured in Ham's F-12 solution (Sigma-Aldrich), 10 % FCS, and 1 % penicillin/streptomycin (PAA). For livecell imaging, the cells were seeded in 35 mm glass-bottom dishes (ibidi, Martinsried, Germany), transfected 24 h later with the indicated constructs using jetPRIME reagent (Polyplus, Illkirch, France) according to the manufacturer's protocols, and further maintained in the same cell culture medium without phenol red. Voltage-clamp measurements with Xenopus oocytes For Xenopus oocyte expression studies, complementary RNA (cRNA) was transcribed in vitro from Nhe-1 linearised (THIK-2) or Xho-1 linearised (THIK-1) plasmids using T7 RNA polymerase (mMessage mMachine T7 Kit, Ambion). cRNA quality was determined by gel electrophoresis and UV spectroscopy. Defolliculated Xenopus oocytes were injected with nuclease-free water containing cRNA (6 ng/oocyte) of wild-type (wt) THIK-1, wt THIK-2, or mutants. Oocytes were incubated at 19 °C for 24–48 h in an ND96 solution containing (mM) 96 NaCl, 2 KCl, 1 MgCl 2 , 1.8 CaCl 2 , and 5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.4–7.5), supplemented with 100 μg/ml gentamycin and 2.5 mM sodium pyruvate. Two-microelectrode voltageclamp measurements with ramp-shaped voltage commands were performed with a TurboTec-10C amplifier
Pflugers Arch - Eur J Physiol
(NPI electronic, Tamm, Germany), and data were recorded at a sampling rate of 120 Hz. The oocytes were placed in a small-volume perfusion chamber and superfused with the ND96 solution. In some of the experiments, the extracellular K+ concentration was elevated to 8, 24, or 96 mM by equimolar replacement of Na+ by K+. For quantification of current amplitude under different experimental conditions, the currents were measured at 0 mV (2 mM K+), −100 mV (24 mM K+), or −80 mV (140 mM K+). All electrophysiological experiments were carried out at room temperature (20–23 °C). To test the effect of halothane on THIK-1 and THIK-2 currents, an appropriate amount of liquid halothane (from Sigma-Aldrich) was injected into the perfusion solution in a gas-tight container (AnaConDa, Sedane Medical) and stirred; the solution was then used immediately for superfusion of the oocytes. Measurement of THIK currents in transfected CHO cells Patch-clamp measurements were performed with CHO cells 24 h after transfection with either wild-type THIK-2, THIK2AAA, THIK-2Δ6-24, or THIK-2AAA/I158G constructs cloned into the pCDNA3.1 mammalian expression vector (Invitrogen). The cells were superfused at room temperature with a bath solution containing (mM) 140 KCl, 0.2 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES; the pH was adjusted to 7.4 with NaOH. Patch-clamp experiments were performed in the whole-cell configuration using pipettes pulled from borosilicate glass capillaries. The patch pipettes (resistance, 3–6 MΩ) were filled with an ‘intracellular’ solution containing (mM) 60 KCl, 65 K-glutamate, 5 EGTA, 3.5 MgCl2, 2 CaCl2, 3 K2ATP, 0.2 Na2GTP, and 5 HEPES; the pH was adjusted to 7.2 with KOH. Steady-state current– voltage relations were obtained by applying slow voltage ramps (40 mV s−1) between −90 and +30 mV. The liquid junction potential between the patch electrode and the bath solution (approximately −8 mV) was not compensated. Live-cell imaging HeLa cells transfected with either enhanced green fluorescent protein (EGFP)-tagged rTHIK-1wt, rTHIK-2wt, rTHIK-2Δ624 , or rTHIK-2AAA constructs were analysed 24–48 h after transfection using an inverted Nikon Eclipse Ti microscope equipped with a ×100 objective (Plan Apo VC ×100 Oil DIC N2, Nikon). Green fluorescent protein (GFP) and DsRed channels were acquired simultaneously using a dualcamera port system composed of custom-made excitation/ emission filters, a dual-band beam splitter and two cooled 14-bit EMCCD cameras (DU-885, Andor Technology). For recording live-cell images, cells were maintained at 37 °C by means of a stage heater (ibidi) with a temperature control system (TC 20, NPI) and an objective heater (PeCon).
Images were acquired and analysed with NIS Elements AR 4 software (Nikon). Statistics Data are reported as means±standard error of the mean (SEM). Statistical significance was determined using Student's t test. In the figures, statistically significant differences to control values are marked by asterisks (*, p
ION CHANNELS, RECEPTORS AND TRANSPORTERS
Breaking the silence: functional expression of the two-pore-domain potassium channel THIK-2 Vijay Renigunta & Xinle Zou & Stefan Kling & Günter Schlichthörl & Jürgen Daut
Received: 8 November 2013 / Accepted: 9 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract THIK-2 belongs to the ‘silent’ channels of the twopore-domain potassium channel family. It is highly expressed in many nuclei of the brain but has so far resisted all attempts at functional expression. THIK-2 has a highly conserved 19-amino-acid region in its N terminus (residues 6–24 in the rat orthologue) that is missing in the closely related channel THIK-1. After deletion of this region (THIK-2Δ6–24 mutant), functional expression of the channel was observed in Xenopus oocytes and in mammalian cell lines. The resulting potassium current showed outward rectification under physiological conditions and slight inward rectification with symmetrical highK+ solutions and could be inhibited by application of halothane or quinidine. Another THIK-2 mutant, in which the putative retention/retrieval signal RRR at positions 14-16 was replaced by AAA, produced a similar potassium current. Both mutants showed a distinct localisation to the surface membrane when tagged with green fluorescent protein and expressed in a mammalian cell line, whereas wild-type THIK2 was mainly localised to the endoplasmic reticulum. These findings suggest that deletion of the retention/retrieval signal RRR enabled transport of THIK-2 channels to the surface membrane. Combining the mutation THIK-2Δ6–24 with a mutation in the pore cavity (rat THIK-2I158G) gave rise to a 12-fold increase in current amplitude, most likely due to an increase in open probability. In conclusion, the characteristics of THIK-2 channels can be analysed in heterologous expression systems by using trafficking and/or gating mutants. The possible mechanisms that enable THIK-2 expression at the surface membrane in vivo remain to be determined.
V. Renigunta (*) : X. Zou : S. Kling : G. Schlichthörl : J. Daut (*) Institute of Physiology and Pathophysiology, Marburg University, Deutschhausstr. 2, 35037 Marburg, Germany e-mail: [email protected] e-mail: [email protected]
Keywords K2P channels . Retention signal . Trafficking . Halothane
Introduction The family of two-pore-domain potassium channels (K2P channels) has fifteen members [7]. Five of these channels, TWIK-1, TWIK-2, KCNK7, TASK-5, and THIK-2, were originally regarded as ‘silent’ channels because no currents associated with these channel proteins could be recorded in native cells and no currents (or only very small currents) could be measured in heterologous expression systems. The reason for the silence of TWIK-1 has recently been detected: It carries an endocytosis signal (DxxxII) that prevents trafficking to the surface membrane. After mutation of the two isoleucine residues, the channel was transported to the surface membrane, and the properties of TWIK-1 currents could be analysed [8]. The currents carried by TWIK-2 [5], KCNK7 [16], and TASK-5 [11] channels are still elusive. In the present study, we tried to elucidate the characteristics of THIK-2 channels [14]. Like all other K2P channels, THIK-2 has two pore domains (P1 and P1) and a long M1–P1 linker. It has a long cytosolic C terminus and a very unusual N-terminal domain containing many proline, arginine, and cysteine residues [14]. We report here that in heterologous expression systems, only a very small fraction of THIK-2 channels is localised to the surface membrane and that THIK-2 channels are efficiently transported to the cell surface when a retention/retrieval signal (RRR) in its N terminus is mutated. Another mutation in the pore domain of THIK-2 appeared to cause an increase in the open probability of the channel. The trafficking mutant and the gating mutants allowed us to describe for the first time the characteristics of THIK-2 channels. Since THIK-2 is highly and differentially expressed in many regions of the brain
Pflugers Arch - Eur J Physiol
[12, 14] as well as in lung, liver, and kidney, it may be assumed that the potassium currents carried by THIK-2 channels are functionally important under some physiological or pathophysiological conditions.
Materials and methods Ethical approval For experiments involving Xenopus oocytes, adult female African clawed frogs (Xenopus laevis) were used. The frogs were anaesthetised by putting them in water containing 1 g/l tricaine. Stage V oocytes were obtained from ovarian lobes. Anaesthesia and operation were carried out in accordance with the principles of German legislation with approval of the animal welfare officer of the Medical Faculty of Marburg University under the governance of the Regierungspräsidium Giessen (the regional veterinary heath authority).
anti-rat immunoglobulin G antibody (Jackson ImmunoResearch) in 1 % BSA/ND96 solution for 60 min. Oocytes were washed thoroughly, initially in 1 % BSA/ND96 (at 4 °C for 60 min) and then in ND96 without BSA (at 4 °C for 15 min). Individual oocytes were placed in 20 μl SuperSignal Elisa Femto solution (Pierce Protein Biology Products, Rockford, IL, USA), and chemiluminescence was quantified in a luminometer (Lumat LB9507, Berthold Technologies). The luminescence produced by un-injected oocytes was used as a reference signal (negative control). To analyse the functionality of the endoplasmic reticulum (ER) localisation signal in the N terminus of THIK-2, we fused the N terminus of THIK-2 with the type 2 protein CD74. The surface expression of this reporter protein construct was determined in COS-7 cells using a luminometric assay described previously [24]. Based on previous experiments with CD74 [17], the N-terminal sequence MHRRRSR was used as a positive control (indicating retention), and CD74 with the mutated N-terminal sequence MHSSSSS was used as a negative control (indicating surface expression).
Molecular cloning and mutagenesis Cell culture Full-length rat THIK-1 (accession no. NM_022293) and rat THIK-2 (accession no. NM_022292) channels cloned into the pSGEM oocyte expression vector were used for voltageclamp experiments [14]. QuikChange (Agilent Technologies) was used to introduce deletions, insertions, and point mutations. For CD74 reporter assays, the N terminus of THIK-2 was fused to the reporter protein CD74. For luminometric measurements of surface expression in Xenopus oocytes, we used a pSGEM vector with THIK-1 or THIK-2 containing an extracellular haemagglutinin (HA) tag (THIK-1 at position 95 and THIK-2 at position 114). The surface membrane marker GFP-C1-PLCδ-PH (the pleckstrin homology domain of phospholipase C delta 1 [20]) was obtained from the non-profit clone repository AddGene (ID: 21179) and later subcloned to the pDsRed-Monomer-C1 vector for membrane co-localisation experiments. All DNA constructs were verified by sequencing. Surface expression assay The surface expression of HA-tagged THIK-1 and THIK-2 channels in Xenopus oocytes was analysed 2 days after injection of cRNA (6 ng/oocyte). Oocytes were incubated for 30 min in an ND96 solution containing 1 % bovine serum albumin (BSA) at 4 °C to block non-specific binding of antibodies. Subsequently, oocytes were incubated for 60 min at 4 °C with 100 μg/ml of rat monoclonal anti-HA antibody (clone 3F10, Roche) in 1 % BSA/ND96, washed six times at 4 °C with 1 % BSA/ND96, and incubated with 0.8 mg/ml peroxidase-conjugated affinity-purified F(ab)2 fragment goat
HeLa and COS-7 cells were cultured in high-glucose DMEM, 10 % foetal calf serum (FCS) (Life Technologies, Paisley, UK) and 1 % penicillin/streptomycin (PAA Laboratories GmbH, Pasching, Austria). Chinese hamster ovary (CHO) cells were cultured in Ham's F-12 solution (Sigma-Aldrich), 10 % FCS, and 1 % penicillin/streptomycin (PAA). For livecell imaging, the cells were seeded in 35 mm glass-bottom dishes (ibidi, Martinsried, Germany), transfected 24 h later with the indicated constructs using jetPRIME reagent (Polyplus, Illkirch, France) according to the manufacturer's protocols, and further maintained in the same cell culture medium without phenol red. Voltage-clamp measurements with Xenopus oocytes For Xenopus oocyte expression studies, complementary RNA (cRNA) was transcribed in vitro from Nhe-1 linearised (THIK-2) or Xho-1 linearised (THIK-1) plasmids using T7 RNA polymerase (mMessage mMachine T7 Kit, Ambion). cRNA quality was determined by gel electrophoresis and UV spectroscopy. Defolliculated Xenopus oocytes were injected with nuclease-free water containing cRNA (6 ng/oocyte) of wild-type (wt) THIK-1, wt THIK-2, or mutants. Oocytes were incubated at 19 °C for 24–48 h in an ND96 solution containing (mM) 96 NaCl, 2 KCl, 1 MgCl 2 , 1.8 CaCl 2 , and 5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.4–7.5), supplemented with 100 μg/ml gentamycin and 2.5 mM sodium pyruvate. Two-microelectrode voltageclamp measurements with ramp-shaped voltage commands were performed with a TurboTec-10C amplifier
Pflugers Arch - Eur J Physiol
(NPI electronic, Tamm, Germany), and data were recorded at a sampling rate of 120 Hz. The oocytes were placed in a small-volume perfusion chamber and superfused with the ND96 solution. In some of the experiments, the extracellular K+ concentration was elevated to 8, 24, or 96 mM by equimolar replacement of Na+ by K+. For quantification of current amplitude under different experimental conditions, the currents were measured at 0 mV (2 mM K+), −100 mV (24 mM K+), or −80 mV (140 mM K+). All electrophysiological experiments were carried out at room temperature (20–23 °C). To test the effect of halothane on THIK-1 and THIK-2 currents, an appropriate amount of liquid halothane (from Sigma-Aldrich) was injected into the perfusion solution in a gas-tight container (AnaConDa, Sedane Medical) and stirred; the solution was then used immediately for superfusion of the oocytes. Measurement of THIK currents in transfected CHO cells Patch-clamp measurements were performed with CHO cells 24 h after transfection with either wild-type THIK-2, THIK2AAA, THIK-2Δ6-24, or THIK-2AAA/I158G constructs cloned into the pCDNA3.1 mammalian expression vector (Invitrogen). The cells were superfused at room temperature with a bath solution containing (mM) 140 KCl, 0.2 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES; the pH was adjusted to 7.4 with NaOH. Patch-clamp experiments were performed in the whole-cell configuration using pipettes pulled from borosilicate glass capillaries. The patch pipettes (resistance, 3–6 MΩ) were filled with an ‘intracellular’ solution containing (mM) 60 KCl, 65 K-glutamate, 5 EGTA, 3.5 MgCl2, 2 CaCl2, 3 K2ATP, 0.2 Na2GTP, and 5 HEPES; the pH was adjusted to 7.2 with KOH. Steady-state current– voltage relations were obtained by applying slow voltage ramps (40 mV s−1) between −90 and +30 mV. The liquid junction potential between the patch electrode and the bath solution (approximately −8 mV) was not compensated. Live-cell imaging HeLa cells transfected with either enhanced green fluorescent protein (EGFP)-tagged rTHIK-1wt, rTHIK-2wt, rTHIK-2Δ624 , or rTHIK-2AAA constructs were analysed 24–48 h after transfection using an inverted Nikon Eclipse Ti microscope equipped with a ×100 objective (Plan Apo VC ×100 Oil DIC N2, Nikon). Green fluorescent protein (GFP) and DsRed channels were acquired simultaneously using a dualcamera port system composed of custom-made excitation/ emission filters, a dual-band beam splitter and two cooled 14-bit EMCCD cameras (DU-885, Andor Technology). For recording live-cell images, cells were maintained at 37 °C by means of a stage heater (ibidi) with a temperature control system (TC 20, NPI) and an objective heater (PeCon).
Images were acquired and analysed with NIS Elements AR 4 software (Nikon). Statistics Data are reported as means±standard error of the mean (SEM). Statistical significance was determined using Student's t test. In the figures, statistically significant differences to control values are marked by asterisks (*, p
