Upregulation of KCNQ1/KCNE1 K⁺ channels by Klotho

Abstract

Klotho is a transmembrane protein expressed primarily in kidney, parathyroid gland, and choroid plexus. The extracellular domain could be cleaved off and released into the systemic circulation. Klotho is in part effective as β-glucuronidase regulating protein stability in the cell membrane. Klotho is a major determinant of aging and life span. Overexpression of Klotho increases and Klotho deficiency decreases life span. Klotho deficiency may further result in hearing loss and cardiac arrhythmia. The present study explored whether Klotho modifies activity and protein abundance of KCNQ1/KCNE1, a K⁺ channel required for proper hearing and cardiac repolarization. To this end, cRNA encoding KCNQ1/KCNE1 was injected in Xenopus oocytes with or without additional injection of cRNA encoding Klotho. KCNQ1/KCNE1 expressing oocytes were treated with human recombinant Klotho protein (30 ng/ml) for 24 h. Moreover, oocytes which express both KCNQ1/KCNE1 and Klotho were treated with 10 µM DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. The KCNQ1/KCNE1 depolarization-induced current (I_Ks) was determined utilizing dual electrode voltage clamp, while KCNQ1/KCNE1 protein abundance in the cell membrane was visualized utilizing specific antibody binding and quantified by chemiluminescence. KCNQ1/KCNE1 channel activity and KCNQ1/KCNE1 protein abundance were upregulated by coexpression of Klotho. The effect was mimicked by treatment with human recombinant Klotho protein (30 ng/ml) and inhibited by DSAL (10 µM). In conclusion, Klotho upregulates KCNQ1/KCNE1 channel activity by 'mainly' enhancing channel protein abundance in the plasma cell membrane, an effect at least partially mediated through the β-glucuronidase activity of Klotho protein.

Introduction

Klotho, a protein expressed in a wide variety of tissues including kidney,^1,2 has a profound impact on aging and life span.^3,4 The extracellular domain of Klotho may function as protease or hormone.⁵ Klotho deficiency results in severe growth retardation and accelerated aging eventually leading to early death.³ Klotho overexpression leads to substantial prolongation of life span.^3,4 Klotho is required for the inhibitory effect of FGF23 on 1,25(OH)₂D₃ producing 1α-hydroxylase.^2,4,6,7 1,25(OH)₂D₃ stimulates intestinal and renal Ca²⁺ and phosphate transport.^8,9 In part due to excessive 1,25(OH)₂D₃ formation, Klotho deficiency increases plasma Ca^{2+ 10} and phosphate⁹ concentration, resulting in vascular calcification,¹¹ growth deficit,² and rapid aging.^2,6,7 Klotho insufficiency further leads to hearing loss, cardiac arrhythmia, and sudden cardiac death.¹ Moreover, Klotho deficiency enhances glucose tolerance.¹²

Hearing loss and cardiac arrhythmia may result from genetic defects of the K⁺ channel subunits KCNE1 or KCNQ1.^13-15 Moreover, KCNQ1 polymorphisms have been associated with diabetes.^16,17 KCNQ1/KCNE1 is expressed in a variety of tissues including the heart,^13,15 skeletal muscle,¹⁸ stria vascularis of the inner ear,¹⁹ renal proximal tubule,²⁰ gastric parietal cells,^21-23 intestinal epithelia,^20,22-26 and hepatocytes.^27-29 KCNQ1 knockout mice suffer from deafness^30,31 and impairment of gastric acid secretion,^31,32 as well as intestinal electrolyte and substrate transport.³³ KCNQ1 deficiency further impairs cell volume regulation.^28,29,34-36 KCNQ1 deficiency further affects cardiac repolarization.³⁷

Besides its impact on 1,25(OH)₂D₃ formation, Klotho may regulate Na⁺, phosphate cotransport,^38,39 Na⁺/K⁺ ATPase,⁴⁰ Ca²⁺ channels⁴¹ and renal outer medullary K⁺ channels⁴² by more direct influence on the channels and transport proteins. The present study thus explored whether Klotho modifies the function of KCNQ1/KCNE1 channels. To this end, voltage-gated current was determined in Xenopus oocytes expressing KCNQ1/KCNE1 with or without coexpression of Klotho, treatment with human recombinant Klotho protein or treatment with DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. Moreover, the effect of Klotho coexpression on KCNQ1/KCNE1 protein abundance at the cell membrane was quantified by chemiluminescence and visualized by confocal microscopy.

Results

The present study explored, whether Klotho modifies KCNQ1/KCNE1 channels. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes without or with cRNA encoding Klotho. In KCNQ1/KCNE1 expressing, but not in water-injected Xenopus oocytes, outward currents (I_Ks) were observed following depolarizing pulses (up to +80 mV), applied from a holding potential of -80 mV (Fig. 1A). Additional expression of Klotho in KCNQ1/KCNE1 expressing oocytes was followed by a significant increase in the amplitude of the peak outward current (I_Ks) at +80 mV (Fig. 1B and C). Plotting the amplitude of the peak outward current (I_Ks) against the corresponding pulse potential revealed the typical slow-delayed activation of KCNQ1/KCNE1 rectifier in the presence and absence of Klotho coexpression (Fig. 1C). Normalization of the peak outward current (I_Ks) to the maximum peak outward current of each respective group dissipated the differences between oocytes coexpressing KCNQ1/KCNE1 with klotho and oocytes expressing KCNQ1/KCNE1 alone (Fig. 1C). Coexpression of Klotho did not significantly modify the KCNQ1/KCNE1 activation threshold. The potential needed to reach the half-maximal peak outward current was similar in Xenopus oocytes expressing KCNQ1/KCNE1 alone and in Xenopus oocytes coexpressing both KCNQ1/KCNE1 and Klotho (Fig. 1D).

Figure 1. Effect of Klotho coexpression on current in KCNQ1/KCNE1 expressing Xenopus oocytes. (A) Original tracings demonstrating outward K⁺ currents activated by depolarization from -120 to +80 mV in 20 mV steps from a holding potential of -80 mV in Xenopus oocytes injected with water (1), injected with cRNA encoding KCNQ1/KCNE1 (2) and in Xenopus oocytes injected with cRNA encoding KCNQ1/KCNE1 and Klotho (3). (B) Arithmetic means ± SEM (n = 16–57) of the normalized depolarization-induced K⁺ current at +80 mV in Xenopus oocytes injected with water (dotted bar), with cRNA encoding KCNQ1/KCNE1(white bar) or with cRNA encoding KCNQ1/KCNE1 and Klotho (black bar). *** indicates statistically significant (P < 0.001) difference of KCNQ1/KCNE1 and Klotho expressing Xenopus oocytes from Xenopus oocytes expressing KCNQ1/KCNE1 alone. (C) Arithmetic means ± SEM (n = 16–57) of the normalized depolarization-induced K⁺ current as a function of voltage in Xenopus oocytes injected with water (gray triangles), with cRNA encoding KCNQ1/KCNE1 (white circles) or with cRNA encoding KCNQ1/KCNE1 and Klotho (black circles). *** indicates statistically significant (P < 0.001) difference of KCNQ1/KCNE1 and Klotho expressing Xenopus oocytes from Xenopus oocytes expressing KCNQ1/KCNE1 alone. (D) Arithmetic means ± SEM (n = 56–57) of the normalized depolarization-induced K⁺ current to the maximum peak current of each respective group as a function of voltage in Xenopus oocytes injected with cRNA encoding KCNQ1/KCNE1 (white circles) or with cRNA encoding KCNQ1/KCNE1 and Klotho (black circles).

Further experiments explored, whether the effect of Klotho coexpression could be mimicked by treatment of KCNQ1/KCNE1 expressing Xenopus oocytes with recombinant human Klotho protein. As shown in Figure 2, treatment of KCNQ1/KCNE1 expressing Xenopus oocytes with recombinant human Klotho protein (30 ng/ml) for 24 h was followed by a significant increase of the KCNQ1/KCNE1 peak outward current (Fig. 2B and C). Similar to what has been observed following coexpressing Klotho in KCNQ1/KCNE1 expressing Xenopus oocytes, the treatment of KCNQ1/KCNE1 expressing Xenopus oocytes with Klotho protein increased the peak outward current values (Fig. 2B and C) but did not significantly modify the KCNQ1/KCNE1 activation threshold (Fig. 2D).

Figure 2. Effect of treatment with recombinant Klotho protein on current in KCNQ1/KCNE1 expressing Xenopus oocytes. (A) Original tracings demonstrating outward K⁺ currents activated by depolarization from -120 to +80 mV in 20 mV steps from a holding potential of -80 mV in Xenopus oocytes injected with water (1),or injected with cRNA encoding KCNQ1/KCNE1 without (2) or with (3) a 24 h pretreatment with recombinant Klotho protein (30 ng/ml). (B) Arithmetic means ± SEM (n = 4–17) of the normalized depolarization-induced K⁺ current at +80 mV in Xenopus oocytes injected with water (dotted bar), or with cRNA encoding KCNQ1/KCNE1 without (white bar) or with (black bar) a 24 h pretreatment with recombinant Klotho protein (30 ng/ml). *** indicates statistically significant (P < 0.001) difference of Klotho treated KCNQ1/KCNE1 expressing Xenopus oocytes from untreated KCNQ1/KCNE1 expressing oocytes. (C) Arithmetic means ± SEM (n = 4–17) of the normalized depolarization-induced K⁺ current as a function of voltage in Xenopus oocytes injected with water (gray triangles), or with cRNA encoding KCNQ1/KCNE1 without (white circles) or with (black circles) a 24 h pretreatment with recombinant Klotho protein (30 ng/ml). *** indicates statistically significant (P < 0.001) difference of Klotho treated KCNQ1/KCNE1 expressing Xenopus oocytes from untreated KCNQ1/KCNE1 expressing Xenopus oocytes. (D) Arithmetic means ± SEM (n = 15–17) of the depolarization-induced K⁺ current (normalized to the maximum peak current of each respective group) as a function of voltage in Xenopus oocytes injected with cRNA encoding KCNQ1/KCNE1 without (white circles) or with (black circles) a 24 h pretreatment with recombinant Klotho protein (30 ng/ml).

Additional experiments were performed to test, whether the effect of Klotho coexpression could be reversed by DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. As illustrated in Figure 3, treatment of Xenopus oocytes expressing both KCNQ1/KCNE1 and Klotho with the DSAL (10 µM) for 48 h significantly blunted the effect of Klotho coexpression on the peak outward current (Fig. 3B and C).

Figure 3. Effect of β-glucuronidase inhibitor (DSAL) on current in KCNQ1/KCNE1 and Klotho expressing Xenopus oocytes. (A) Original tracings demonstrating outward K⁺ currents activated by depolarization from -120 to +80 mV in 20 mV steps from a holding potential of -80 in Xenopus oocytes injected with water (1), injected with cRNA encoding KCNQ1/KCNE1 alone (2) and in Xenopus oocytes injected with cRNA encoding both KCNE1/KCNQ1 and Klotho in the absence of β-glucuronidase inhibitor (3), or in the presence of DSAL for 24 h (4) or 48 h (5). (B) Arithmetic means ± SEM (n = 9–23) of the normalized depolarization-induced K⁺ current at +80 mV in Xenopus oocytes injected with water (dotted bar), injected with cRNA encoding KCNQ1/KCNE1 alone (white bar) or injected with cRNA encoding both KCNQ1/KCNE1 and Klotho in the absence of β-glucuronidase inhibitor DSAL (black bar), or in the presence of DSAL for 24 h (first gray bar) or 48 h (second gray bar). ** indicates statistically significant (P < 0.01) difference of Klotho and KCNQ1/KCNE1 expressing Xenopus oocytes from Xenopus oocytes expressing KCNQ1/KCNE1 alone. ## indicates statistically significant (P < 0.01) difference of DSAL-treated from untreated KCNQ1/KCNE1 and Klotho expressing Xenopus oocytes. (C) Arithmetic means ± SEM (n = 9–23) of the normalized depolarization-induced K⁺ current as a function of voltage in Xenopus oocytes injected with water (gray triangles), injected with cRNA encoding KCNQ1/KCNE1 alone (white circles) and in Xenopus oocytes injected with cRNA encoding both KCNQ1/KCNE1 and klotho in the absence of β-glucuronidase inhibitor DSAL (black circles), or in the presence of DSAL for 48 h (black triangles). ** indicates statistically significant (P < 0.01) difference of Klotho and KCNQ1/KCNE1 expressing Xenopus oocytes from Xenopus oocytes expressing KCNQ1/KCNE1 alone. (D) Arithmetic means ± SEM (n = 18–23) of the depolarization-induced K⁺ current (normalized to the maximum peak current of each group) as a function of voltage in Xenopus oocytes injected with cRNA encoding KCNQ1/KCNE1 and Klotho without treatment (white circles) or treated for 48 h with DSAL (black circles).

In a final series of experiments chemiluminescence and confocal microscopy were employed to test, whether Klotho coexpression influenced the KCNQ1/KCNE1 protein abundance in the cell membrane. As illustrated in Figure 4B, coexpression of both Klotho and KCNQ1-Flag/KCNE1 was followed by a significant increase of KCNQ1-Flag/KCNE1 abundance in the plasma membrane as determined by chemiluminescence. The same effect was also visualized by confocal microscopy (Fig. 4A). The coexpression of Klotho thus increased KCNQ1/KCNE1 channel protein abundance in the plasma membrane.

Figure 4. Effect of Klotho coexpression on KCNQ1/KCNE1 protein abundance in the cell membrane of Xenopus oocytes. (A) Confocal microscopy of the KCNQ1/KCNE1 protein abundance in Xenopus oocytes injected with water (left), injected with cRNA encoding KCNQ1/KCNE1 alone (middle) or expressing KCNQ1/KCNE1 together with Klotho (right). The images are representative for 3 independent experiments. (B) Arithmetic means ± SEM (n = 55–76) of the chemiluminescence of KCNQ1-Flag/KCNE1 protein abundance in Xenopus oocytes injected with water (dotted bar), injected with cRNA encoding KCNQ1-Flag/KCNE1 alone (white bar), or expressing KCNQ1-Flag/KCNE1 with Klotho (black bar). * (P < 0.05) indicates statistically significant difference from the protein abundance in Xenopus oocytes expressing KCNQ1-Flag/KCNE1 alone.

The effect of Klotho coexpression could again be mimicked by treatment of KCNQ1/KCNE1 expressing Xenopus oocytes with recombinant human Klotho protein. As shown in Figure 5, treatment of KCNQ1/KCNE1 expressing Xenopus oocytes with recombinant human Klotho protein (30 ng/ml) for 24 h was followed by a significant increase of the KCNQ1/KCNE1 KCNQ1-Flag/KCNE1 abundance in the plasma membrane).

Figure 5. Effect of treatment with recombinant Klotho protein on KCNQ1/KCNE1 protein abundance in the cell membrane of Xenopus oocytes. Arithmetic means ± SEM (n = 70–72) of the chemiluminescence of KCNQ1-Flag/KCNE1 protein abundance in Xenopus oocytes injected without (dotted bar) or with cRNA encoding KCNQ1-Flag/KCNE1 without (white bar) or with (black bar) a 24 h pretreatment with recombinant Klotho protein (30 ng/ml). *** (P < 0.001) indicates statistically significant difference from the protein abundance in Xenopus oocytes expressing KCNQ1-Flag/KCNE1 alone.

Discussion

The present study uncovers a novel function of Klotho, i.e., the upregulation of the slowly activating outward current generated by the heterotetrameric K⁺ channel KCNQ1/KCNE1. Coexpression of Klotho or treatment with the Klotho protein increased the abundance of channel protein and the respective K⁺ conductance. The effect may be due to direct influence of Klotho on the channel protein or due to influence of other oocyte molecules indirectly modifying KCNQ1/KCNE1 protein abundance in the cell membrane. Klotho may affect primarily the KCNE1 subunit, the KCNQ1 subunit or both ion channel subunits in parallel.

In the heart, stimulation of KCNQ1 is expected to accelerate repolarization, whereas KCNQ1 inhibition delays cardiac repolarization.⁴³ KCNE1 determines the activation time course of the heterotetrameric channel.⁴⁴ KCNE1/KCNQ1 channel activity is thus decisive for cardiac function.^13-15 At least in theory, decreased stimulation of KCNQ1 in the heart could contribute to the occurrence of cardiac arrhythmia in Klotho hypomorphic mice.¹

K⁺ channel activity is further a determinant of tubular transport. In the proximal renal tubule K⁺ channels provide the driving force for Na⁺-coupled transport of glucose and other substrates across the apical membrane and at the same time decreases electrogenic HCO₃^- exit across the basolateral cell membrane, thus influencing cytosolic pH and apical Na⁺/H⁺ exchanger.⁴⁵ Accordingly, proximal renal tubular transport is compromised in animals lacking KCNQ1.²⁰

KCNQ1 is further expressed in liver,^27-29 skeletal muscle,¹⁸ and several epithelia.^21-26,33 In the liver, for instance, KCNQ1 governs cell volume and thus cell volume-sensitive functions including glucose uptake.⁴⁶ Beyond that KCNQ1 is important for a variety of functions including hearing,^30,31 gastric acid secretion,^31,32 as well as intestinal and renal transport.³³

K⁺ channels are decisive for cell volume regulation.^47,48 K⁺ channels further influence cellular K⁺ loss during apoptosis and thus participate in the machinery of suicidal cell death.^49-53 By influencing HCO₃^- exit K⁺ channel activity influences cytosolic pH, which in turn influences caspase activation⁵⁴ and glycolysis.⁵⁵

In conclusion, the present observations point to a novel effect of Klotho, i.e., the upregulation of the slowly activating heterotetrameric K⁺ channel KCNQ1/KCNE1. At least in theory, loss of this effect may contribute to the consequences of Klotho deficiency.

Materials and Methods

Xenopus Oocytes were explanted from adult Xenopus laevis (NASCO). Xenopus laevis frogs were anesthesized by a 0.1% Tricain solution. After confirmation of anesthesia and disinfection of the skin, a small abdominal incision was made and oocytes were removed, followed by closure of the skin by sutures. All animal experiments were conducted in accordance with the Helsinki Declaration of 1975 and according to the German law for the welfare of animals and the surgical procedures on the adult Xenous laevis were reviewed and approved by the respective government authority of the state Baden-Württemberg (Regierungspräsidium) prior to the start of the study (Anzeige für Organentnahme nach §6).

Constructs

For generation of cRNA, constructs were used encoding wild-type human KCNQ1/KCNE1⁵⁶ wild-type human KCNQ1-Flag carrying an extracellular Flag tag epitope⁵⁷ and wild-type mouse Klotho.³⁸ The constructs were used for the generation of cRNA as described previously.^58,59

Voltage clamp in Xenopus oocytes

Xenopus oocytes were prepared as previously described.^60,61 cRNA encoding KCNQ1 (3.5 ng) and 1.5 ng cRNA encoding KCNE1 were injected with or without 10 ng of cRNA encoding Klotho⁶² on the next day of preparation of the Xenopus oocytes. All experiments were performed at room temperature 3 d after injection.^63,64 The oocytes were maintained at 17 °C in ND96 solution containing: 88.5 mM NaCl, 2 mM KCl, 1 mM MgC1₂, 1.8 mM CaC1₂, 5 mM HEPES, Tretracycline (Sigma, 0.11 mM), Ciprofloxacin (Sigma, 4 μM), Gentamycin (Refobacin, 0.2 mM) and Theophylin (Euphylong, 0.5 mM) as well as Sodium Pyruvate (Sigma, 5 mM) were added to the ND96, pH was adjusted to 7.5 by addition of NaOH. The control superfusate (ND96) contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂,1 mM MgCl₂ and 5 mM HEPES, pH was adjusted to 7.4 by addition of NaOH. Where indicated, recombinant human β-Klotho protein (10, 30 or 50 ng/ml, R&D Systems) and D-saccharic acid 1,4-lactone monohydrate (DSAL, 10 µM, Sigma) were added. In 2-electrode voltage-clamp experiments KCNQ1/KCNE1 channel currents were elicited every 10 s with 3 s depolarizing pulses up to +80 mV applied from a holding potential of -80 mV. Pulses were applied in 20 mV increments. The data were filtered at 2 kHz and recorded with a Digidata 1322A A/D-D/A converter and ClampexV 0.9.2 software for data acquisition (Axon Instruments).^65,66 The analysis of the data was performed with Clampfit 9.2 (Axon Instruments) software.

Chemiluminescence

For detection of KCNQ1-Flag cell surface expression, the oocytes were first incubated with primary monoclonal mouse anti-Flag antibody (1:200, Sigma Aldrich) and subsequently with secondary, HRP-conjugated anti-mouse IgG antibody (1:2500, GE Healthcare Life Sciences). Individual oocytes were placed in 96 well plates with 20 µl of SuperSignal ELISA Femto Maximum Sensitivity Substrate (Pierce) and chemiluminescence of single oocytes was quantified in a luminometer (Walter Wallac 2 plate reader, Perkin Elmer) by integrating the signal over a period of 1 s. Results display normalized relative light units. Integrity of the measured oocytes was assessed by visual control after the measurement to avoid unspecific light signals from the cytosol.^64,67

Immunocytochemistry

To visualize KCNQ1 cell surface expression the oocytes were fixed in 4% paraformaldehyde for 2 h at room temperature. After washing with PBS, the oocytes were cryoprotected in 30% sucrose, frozen in mounting medium and placed on cryostat. Sections were collected at a thickness of 8 µm on coated slides and stored at -20 °C. For immunostaining, sections were dried at room temperature, fixed in aceton/methanol (1:1), washed in PBS and blocked for 1 h in 5% bovine serum albumin in PBS. The primary antibody (rabbit polyclonal directed to the KCNQ1-Carboxyterminal end, 1:250, Abcam) was incubated overnight at 4 °C. Binding of primary antibody was visualized with a goat anti-rabbit-FITC conjugated IgG antibody (1:1000, Invitrogen, Molecular Probes). Then, oocytes were analyzed by a fluorescence laser scanning microscope (LSM 510, Carl Zeiss MicroImaging GmbH) with A-Plan 40x/0.25.⁶⁸ Brightness and contrast settings were kept constant during imaging of all oocytes in each injection series.

Statistical analysis

Data are provided as means ± SEM, n represents the number of experiments. All oocyte experiments were repeated with at least 3 batches of oocytes; in all repetitions qualitatively similar data were obtained. Data were tested for significance using ANOVA 1-way, and results with P < 0.05 were considered statistically significant.

10.4161/chan.27662