Doi:10.1016/j.bbrc.2007.12.168Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 367 (2008) 687–692 Lysosome mediated Kir2.1 breakdown directly inﬂuences inward rectiﬁer current density John A. Jansen, Teun P. de Boer, Rianne Wolswinkel, Toon A.B. van Veen, Marc A. Vos, Harold V.M. van Rijen, Marcel A.G. van der Heyden * Department of Medical Physiology, Division Heart & Lungs, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands Received 18 December 2007 Available online 7 January 2008 The inward rectiﬁer current generated by Kir2.1 ion channel proteins is primarily responsible for the stable resting membrane poten- tial in various excitable cell types, like neurons and myocytes. Tight regulation of Kir2.1 functioning prevents premature action potentialformation and ensures optimal repolarization times. While Kir2.1 forward traﬃcking has been addressed in a number of studies, its deg-radation pathways are thus far unknown. Using three diﬀerent lysosomal inhibitors, NH4Cl, chloroquine and leupeptin, we now dem-onstrate involvement of the lysosomal degradation pathway in Kir2.1 breakdown. Upon application of the inhibitors, increased steadystate protein levels are detectable within few hours coinciding with intracellular granular Kir2.1 accumulation. Treatment for 24 h witheither chloroquine or leupeptin results in increased plasmamembrane originating inward rectiﬁer current densities, while current–voltagecharacteristics remain unaltered. We conclude that the lysosomal degradation pathway contributes to Kir2.1 mediated inward rectiﬁercurrent regulation.
Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Inward rectiﬁer; Kir2.1; Lysosome; Chloroquine; Degradation The formation of an action potential (AP) stands at the izing currents) and ﬁne tuning of ﬁnal repolarization basis of many physiological processes. A number of dis- (directly, contributing repolarizing current) of the AP tinct diseases of the cardiovascular system (atrial or ven- In mammals, several diﬀerent but closely related ion chan- nel proteins constitute the cardiac ventricular IK1 channel.
migraine) and the motor system (myotonia) can be related Of these, the KCNJ2 and KCNJ12 gene products Kir2.1 to ion channel malfunctioning The AP of excitable and Kir2.2 are the main determinants. To function as an cells, such as neurons and myocytes, is the resultant of ion channel, Kir2.x proteins form either homotypic or het- sequential and coordinated activity of a number of ion erotypic tetramers deﬁned by speciﬁc sequence domains channels. Depolarizing currents are generally carried by Several studies indicate that manipulating IK1 by means of sodium and calcium ions, while repolarizing currents result null mutation overexpression or dominant nega- mainly from potassium ﬂuxes. The inward rectiﬁer potas- tive expression of Kir2.1 elucidates the importance sium current (IK1) is one of the few that operates between of Kir2.1 mediated IK1 for normal AP formation and con- subsequent APs and is primarily responsible for generating trol of sinus rhythm. The ultimate expression level of and stabilizing the resting membrane potential at a rather Kir2.1 may inﬂuence the eventual arrhythmogenic out- negative level between 90 mV, and secondly come. In humans, loss-of-function mutations in Kir2.1 lead for the initial depolarization (indirectly, opposing depolar- to Andersen–Tawil syndrome which is characterized bypotentially lethal ventricular arrhythmias, periodic paraly- sis and dysmorphic features, further emphasizing the pleio- Corresponding author. Fax: +31 30 2539036.
tropic action of Kir2.1 in development and adult E-mail address: (M.A.G. van der physiology .
0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2007.12.168 J.A. Jansen et al. / Biochemical and Biophysical Research Communications 367 (2008) 687–692 Traﬃcking of potassium channels involves a large num- PAGE and blotted onto nitrocellulose membrane (Bio-Rad, Veenendaal, ber of subsequent steps, all of which are subject to tight reg- The Netherlands). Blots were incubated with GFP (cat. no. Sc-9996; SantaCruz Biotechnology, Santa Cruz, CA, USA) and Pan-Cadherin (cat. no.
ulation . Several elegant studies have addressed C1821; Sigma) primary antibodies and peroxidase-conjugated secondary molecular mechanisms of Kir2.x traﬃcking towards and antibody (Jackson ImmunoResearch, West Grove, PA, USA). Standard anchoring at the plasma membrane. This disclosed several ECL procedure was used as ﬁnal detection (Amersham Bioscience, Kir2.x intracellular N- and C-terminal domains and inter- acting proteins involved in endoplasmic reticulum retention, Immunoﬂuorescence microscopy. HEK-KWGF or HEK-HsKir2.1 cells cultured on Ø 11 mm glass cover slips (Smethwick, Warley, UK) were ﬁxed forward traﬃcking and plasma membrane targeting using 4% paraformaldehyde. After permeabilization with 0.2% Triton X- Several human KCNJ2 mutations that display traﬃcking 100 (BDH) in PBS, cells were pre-incubated with 2% BSA. Next, cells were defects have been identiﬁed , but interestingly these incubated overnight with either anti-GFP or anti-Kir2.1 (cat. no. Sc-18708; mutations are not in the previously mentioned export signal Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibody for sequences. Finally, celastrol, a bioactive compound with HEK-KWGF and HEK-HsKir2.1 cells, respectively, followed by incuba-tion with anti-mouse or anti-goat FITC conjugated secondary antibody strong antioxidant characteristics, inhibits Kir2.1 traﬃcking (Jackson ImmunoResearch) for 2 h. Cover slips were mounted with Vec- towards the plasmamembrane in HEK293 cells As tashield (Vector Laboratories Inc., Burlingame, CA, USA) and imaged demonstrated by Tong et al., tyrosine242 of Kir2.1 is using a Nikon Optiphot-2 microscope equipped for epiﬂuorescence.
involved in clathrin mediated endocytosis, by a tyrosine Electrophysiology. IK1-currents were recorded using the whole cell kinase dependent mechanism . However, beyond endo- voltage clamp conﬁguration in randomly chosen single HEK-KWGF cellsusing an Axopatch 200B ampliﬁer (Molecular Devices, Toronto, Canada).
cytosis no experimental evidence has been described on the Currents were low-pass ﬁltered at 2 kHz and recorded at 4 kHz using a pathways involved in Kir2.x degradation thus far. In this Apple PowerMac ﬁtted with A/D card (National Instruments, Austin, study we focused on the lysosomal degradation pathway TX, USA). From a holding potential of 40 mV, 750 ms long square test by using three diﬀerent inhibitors of lysosomal breakdown pulses to potentials ranging between 100 and +50 mV were applied to . The lysosomal degradation pathway starts in endo- elicit membrane currents. Steady state currents at the end of the test pulsewere normalized to membrane capacitance and plotted versus test somes containing trapped membrane proteins, followed by potential. To obtain membrane conductances, the slope of I–V plot fusion with early lysosomes which results in mature lyso- 80 mV was determined using linear regression.
somes (for reviews see ). The acid environment in Experiments were done at 20 °C using an extracellular solution con- the lysosomes is required for protein digestion by acidic taining (mmol/L) 140 NaCl, 17.5 NaCO3, 15 Hepes, 6 glucose, 5.4 KCl, hydrolases. We used the lysosomal protease inhibitors chlo- 1.8 CaCl2 and 1 MgCl2 in H2O (pH 7.20, NaOH). Patch pipettes wereﬁlled with an internal solution containing (mmol/L) 125 potassium glu- roquine, ammoniumchloride (NH4Cl) and leupeptin. Chlo- conate, 10 KCl, 5 Hepes, 5 EGTA, 4 Na2ATP, 2 MgCl2 and 0.6 CaCl2 in roquine, an antimalarial drug, and NH4Cl are weak bases, H2O (pH 7.20, KOH) and had resistances ranging between 2 and 5 MX.
increasing the lysosomal pH and thereby prohibiting the Liquid junction potential was calculated using pClamp (Molecular Devi- breakdown of proteins by hydrolases. Furthermore, both ces) and used for oﬄine correction.
chemicals inhibit the transport of hydrolases to the lyso- Statistics. All data are presented as mean ± SEM. Diﬀerences among groups were evaluated using one-way ANOVA and a post-hoc Holm– somes. Leupeptin, on the other hand, acts directly as an Sidak test, signiﬁcance was assumed if p < 0.05.
inhibitor of the hydrolases. Interestingly, leupeptin appearsto aﬀect only the lysosomes, whereas NH4Cl and chloro-quine inﬂuence both the lysosomes and the endosomes. In this study we demonstrate that inhibition of lysosomal pro-tein breakdown results in increased steady state level and Inhibition of lysosomal breakdown pathways increases Kir2.1 intracellular accumulation of Kir2.1 protein, and elevated steady state levels IK1 current densities.
HEK-KWGF cells were incubated with either 1 mM NH4Cl, 10 lM chloroquine or 5 lg/mL leupeptin for 6 Materials and methods and 24 h, respectively. Subsequently, expression level ofKir2.1–GFP fusion protein was analyzed by Western blot- Cell culture and pharmacological treatment. HEK-KWGF cells stably ting. As depicted in A, chloroquine rapidly increased expressing wildtype murine Kir2.1–GFP fusion protein were generatedand cultured as described previously . HEK-HsKir2.1 cells stably Kir2.1–GFP expression levels within 6 h which was even express non-tagged human Kir2.1 from a pcDNA3 (InVitrogen, Breda, more pronounced after 24 h. Leupeptin resulted in The Netherlands) based expression vector. For lysosomal degradation enhanced Kir2.1–GFP following 6 and 24 h of incubation.
pathway inhibition, NH4Cl (BDH, Poole, UK), chloroquine (Sigma, St.
Only modest increased Kir2.1–GFP levels were observed Louis, MO, USA) and leupeptin (Sigma) were added to the culture medium to obtain ﬁnal concentrations of 1 mM, 10 lM and 5 lg/mL, 4Cl application. Following the strong upregulation respectively for the indicated time at the end of the experiment until due to chloroquine or leupeptin treatment, an additional harvesting or electrophysiological recording.
signal is observed at 70 kDa. Since the fusion protein is Western blotting. HEK-KWGF cells were lysed in RIPA buﬀer detected by an antibody directed against the C-terminal (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 10 mM Na2HPO4, 1% (v/v) fused GFP, this product most likely represents a protein Triton X-100, 1% (w/v) Na-deoxycholate, 0.1% (w/v) SDS, 1mM EDTA, product that results from N-terminal Kir2.1 cleavage. To 50 mM NaF, 2 mM PMSF and 14 lg/ml aprotinin). Lysates were clariﬁedby centrifugation at 14,000 rpm for 10 min at 4 °C and mixed with loading further elucidate the kinetics of Kir2.1 upregulation, buﬀer. Twenty micrograms of proteins were separated by 10% SDS– HEK-KWGF cells were treated with 10 lM chloroquine
J.A. Jansen et al. / Biochemical and Biophysical Research Communications 367 (2008) 687–692 for 1, 2, 3, 4, 6 and 24 h, respectively (B). Increasedexpression levels of full-length Kir2.1–GFP were observedalready after 1 h of treatment. Furthermore, the presumed70 kDa degradation product becomes detectable follow-ing 3 h of chloroquine treatment. These data indicate thatthe lysosomal degradation pathway is involved in Kir2.1–GFP breakdown, and that Kir2.1–GFP turnover takesplace at a time scale of only a few hours.
Lysosomal inhibition results in granular intracellular Kir2.1accumulation Next, we assessed Kir2.1–GFP localization following application of the diﬀerent inhibitors for 6 and 24 h. Innon-treated cells, Kir2.1–GFP is mainly localized at theplasmamembrane. Chloroquine incubation leads to strongintracellular accumulation A). Similar results wereobtained using non-tagged Kir2.1 Like for chlo-roquine, incubation with leupeptin or NH4Cl results inintracellular accumulation of Kir2.1–GFP and appearedto increase plasmamembrane staining (We con-clude that the strong increase in Kir2.1–GFP levels in chlo-roquine, and to a lesser extend leupeptin and NH4Cl,treated cells substantially results from intracellular granu-lar accumulation, presumably in late endosomes and/orlysosomes .
Fig. 2. Intracellular Kir2.1 accumulation upon treatment of HEK- Hrs 10 μM Chloroquine
KWGF or HEK-HsKir2.1 cells with lysosomal inhibitors. (A) Cellcultures were treated as in . Subsequently, Kir2.1–GFP was detected in ﬁxed cells using anti-GFP as primary and Fitc-labeled secondaryantibody. Scale bar represents 40 lm. (B) Intracellular accumulation ofnon-tagged Kir2.1 (green) upon treatment of HEK-HsKir2.1 cells with 10 lM chloroquine for 2 and 4 h, respectively. Kir2.1 was detected by anti-Kir2.1 antibody, nuclei are stained with DAPI (blue). Scale bar represents Fig. 1. Eﬀect of lysosomal degradation inhibitors on Kir2.1–GFP steady 20 lm. (For interpretation of the references in color in this ﬁgure legend, state expression levels. (A) Kir2.1–GFP protein expression levels in HEK- the reader is referred to the web version of this article.) KWGF cells increase upon treatment with diﬀerent lysosomal inhibitors.
Inhibition of lysosomal mediated Kir2.1 breakdown results in HEK-KWGF cell cultures were treated with 1 mM NH4Cl, 10 lMchloroquine or 5 lg/mL leupeptin for 6 and 24 h, respectively. Kir2.1– increased IK1 densities GFP protein level in total cell lysates was detected using GFP antibody.
(B) Kinetics of Kir2.1–GFP upregulation following treatment with 10 lM To see whether the increased protein levels as observed chloroquine. Cadherin expression levels were identical to total protein by Western blotting and intracellular accumulation would proﬁles as detected by Ponceau S staining prior to immunodetection (not also result in enhanced functional Kir2.1–GFP expression shown), and are regarded as loading control using a pan-cadherinantibody.
at the plasmamembrane, HEK-KWGF cells treated with J.A. Jansen et al. / Biochemical and Biophysical Research Communications 367 (2008) 687–692 chloroquine or leupeptin were analyzed for IK1 current plasmamembrane. Apparently, our 24 h treatment with densities (As depicted in A and B, chloro- chloroquine and leupeptin results in saturation of the quine signiﬁcantly increased the inward component of IK1 Kir2.1 degradation pathway which likely aﬀects internali- at membrane potentials between zation capacity of the functional Kir2.1 channels, culmi- both chloroquine and leupeptin signiﬁcantly increased the nating in increased IK1 densities. Preliminary studies outward component of IK1 between membrane potentials indicated that NH4Cl did not result in a signiﬁcant increase 65 mV Furthermore, the reversal in IK1 densities, which is in line with the biochemical potential was not changed, which was reﬂected by a lack results. Furthermore, chloroquine and NH4Cl display the same mechanism of action with respect to lysosomal degra- 74.9 ± 0.6 mV for control, chloroquine dation inhibition although with diﬀerent eﬃciencies. In and leupeptin, respectively).
contrast to our results, transiently transfected Kir6.2 chan-nels in COS cells do not display increased cell surface expression upon 6 or 12 h of chloroquine treatment .
This may be due to diﬀerent methodology between their In the current study we provide biochemical, immuno- (chemiluminescence) and our (patch clamp) study to detect ﬂuorescence and electrophysiological evidence for involve- ion channel expression at the plasmamembrane. On the ment of the lysosomal degradation pathway in Kir2.1 other hand, Kir6.2 channels may be degraded by a diﬀerent breakdown. Results of Tong et al. suggest that pathway or multiple pathways, or at a diﬀerent time scale.
Kir2.1 channels enter the degradation pathway via clathrin Finally, Kv1.5 voltage gated channel is degraded via the mediated endocytosis. Whether all Kir2.1 channels are sub- proteasomal pathway instead of the lysosomal pathway sequently degraded via the lysosomal pathway is unknown.
and blocking this degradation pathway results in increased Alternatively, internalized channels may become targeted IKur current densities Obviously, no universal potas- to proteasome mediated degradation or recycle to the sium ion channel protein degradation pathway seems to increased steady state expression levels within 1 h of treat- Chloroquine and leupeptin treatment yields a product ment with lysosomal inhibitors (B). This implicates a that is approximately 10 kDa less in molecular weight relatively rapid turnover, within hours, of the Kir2.1 ion than full-length Kir2.1–GFP. Since GFP is fused to the channel protein in HEK293 cells. Furthermore, lysosomal C-terminus of Kir2.1, we reason that this product is inhibition results in intracellular Kir2.1 protein accumula- the result of N-terminal cleavage. It might have a much tion as depicted in within 2 h, however this does not shorter half-life than full length protein and therefore necessarily imply a retarded internalization from the only becomes detectable following maximal inhibition Fig. 3. Quantiﬁcation of functional membrane expression of Kir2.1 using the whole cell voltage clamp technique. (A) Diagram depicting the relationbetween steady state IK1 current and test potential. Compared to controls, inward IK1 currents are larger in chloroquine and leupeptin treated HEK-KWGF cell cultures. Asterisk indicates a signiﬁcant diﬀerence between control and chloroquine (p < 0.05). (B) Steady state conductance density negativeto Ek were signiﬁcantly increased in chloroquine treated cultures, underscoring increased membrane localization of Kir2.1 channels (p < 0.05). (C) Detailof outward currents depicted in (A); legend as in (A). Both chloroquine and leupeptin treated cultures show signiﬁcantly increased outward current (* and#, p < 0.05).
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Psychiatrist Beats Suit Blaming Him for Patient's Killing Mom Milton Satcher and Laura Strong defended a psychiatrist accused of breaching the standard of care bycutting a patient's medications.John Disney/Daily Report After 10 years and two appeals, the guardians of a mental y disabled man who kil ed his mother weeks after hisdoctor took him off anti-psychotic medications have walked away with nothing from their medical malpractice suit.
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