Cardiovascular Research

Cardiovascular Research 2003 59(2):328-338; doi:10.1016/S0008-6363(03)00366-3
© 2003 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Schram, G. Articles by Nattel, S. Search for Related Content PubMed PubMed Citation Articles by Schram, G. Articles by Nattel, S. Social Bookmarking          What's this?

Copyright © 2003, European Society of Cardiology

Barium block of Kir2 and human cardiac inward rectifier currents: evidence for subunit-heteromeric contribution to native currents

Gernot Schram^a,b, Marc Pourrier^a,d, Zhiguo Wang^a,b, Michel White^a,b and Stanley Nattel^a,b,c,*

^aDepartment of Medicine and Research Center, Montreal Heart Institute, 5000 Belanger Street East, Montreal, Quebec, Canada, H1T 1C8
^bDepartment of Medicine, University of Montreal, Montreal, Quebec, Canada
^cDepartment of Pharmacology, McGill University, Montreal, Quebec, Canada
^dDepartment of Pharmacology, University of Montreal, Montreal, Quebec, Canada

^* Corresponding author. Tel.: +1-514-376-3330; fax: +1-514-376-1355. nattel{at}icm.umontreal.ca

Received 14 January 2003; accepted 27 March 2003

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
Background: Kir2 subunits are believed to underlie the cardiac inwardly rectifying current I_K1. The subunit composition of native I_K1 currents is uncertain, and it has been suggested that heteromultimer formation may play a role. Methods: We studied Ba²⁺ block of homo- and heteromeric Kir2 channels in Xenopus oocytes and compared the properties observed to those of human cardiac I_K1 in cells isolated from myocardial biopsies of normal human hearts. Results: Homomeric expression of Kir2.1 and Kir2.3 produced currents with similar Ba²⁺ sensitivities (e.g. IC₅₀ at –120 mV: 16.2±3.4, n = 11 and 18.5±2.1, n = 10, respectively), but these were less sensitive to Ba²⁺ than native I_K1 (4.7±0.5 µM, n = 10, P = 0.001, P<0.001, respectively) and had different Ba²⁺ blocking kinetics from cardiac I_K1. Kir2.2 sensitivity was similar to cardiac I_K1 (e.g., 2.8±0.4 µM, Kir2.2, n = 9, vs. 4.7±0.5 µM for I_K1), but the blocking kinetics of Kir2.2 were faster than those of I_K1. Currents resulting from co-expression of Kir2 subunits had similar Ba²⁺ sensitivities and blocking kinetics among groups and were similar to I_K1 in both Ba²⁺ sensitivity (e.g., IC₅₀ at –120 mV: 4.5±1.0, 2.5±0.5, and 2.3±0.4 µM for co-injected Kir2.1/2.2, n = 6, Kir2.1/2.3, n = 5, and Kir2.2/2.3, n = 4, respectively) and blocking kinetics. Conclusion: Co-injection of Kir2 subunits results in currents with Ba²⁺ blocking properties different from homomeric Kir2 expression but similar to cardiac I_K1. These observations suggest that a substantial proportion of native I_K1 may result from heteromultimer formation among diverse Kir2 family subunits.

KEYWORDS Ion transport; K-channel; Arrhythmia (mechanisms); Ion channels; Sudden death

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
The inward rectifier current (I_K1) was first described in 1949 in skeletal muscle [1]. In the heart, I_K1 sets the resting potential close to the K⁺ equilibrium potential [2] and contributes significantly to cardiac repolarization [3–5]. Dysfunction of I_K1 causes cardiac arrhythmias in Andersen's syndrome (LQT7) [6].

A variety of genes encoding inwardly rectifying K⁺ (Kir) channel subunits has been cloned and grouped into seven families (Kir1–7). Subunits of the Kir2 family are believed to underlie I_K1 in the heart [2]. Three pore-forming -subunits of the Kir2 family, Kir2.1–3, are expressed in cardiomyocytes [7]. In the human heart, Kir2.1 transcripts are 10-fold more abundant than those of Kir2.2 or Kir2.3 [8]. The relative expression of Kir2 transcripts fails to explain a variety of cardiac I_K1 properties [8]. Heteromultimer formation among Kir2 subunits results in currents distinct from those produced by homomeric Kir2 subunit expression [9,10], potentially contributing to phenotypic diversity in Andersen's syndrome [9]. However, no study in the literature has compared directly currents carried by homomeric and heteromeric Kir2 channels to native I_K1.

High-potency block by extracellular Ba²⁺ is a striking pharmacological property of I_K1 [10]. Kir2-based channels have different sensitivities to Ba²⁺ [7,9–11]. The goal of the present study was to compare Ba²⁺-blocking properties of currents carried by single Kir2 subunits with currents carried by co-expressed subunits and with human cardiac I_K1.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
2.1 Functional expression of cloned inward rectifier subunits in Xenopus oocytes
Kir2.1 and Kir2.2 were subcloned into a modified expression vector (pCRII, Invitrogen) containing a poly (A+) tail (A+-pCRII). Kir2.1-A+-pCRII was a kind gift of Lily Leh Jan, San Francisco, CA, USA. Kir2.2-A+-pCRII was a kind gift of Barbara Wible, Cleveland, OH, USA. After linearization with BamHI, cRNA for injection into Xenopus oocytes was prepared with T7 RNA polymerase. Kir2.3 was a kind gift of Carol Vandenberg, Santa Barbara, CA, USA. Kir2.3 cDNA was subcloned into a Bluescript SK- (Stratagene) vector. After linearization with HindIII, cRNA was prepared with T3 RNA polymerase, and 6 to 12 ng cRNA of either a single clone or a mixture of equal amounts of different cRNAs (two at a time) to a total amount of 6 to 12 ng were injected into stage IV–V Xenopus oocytes, followed by two-electrode voltage-clamp 24–72 h later. Currents were elicited at 22°C by 750-ms voltage steps from a holding potential of –60 mV to test potentials between –150 mV and +30 mV in 10 mV increments with a GeneClamp-500 amplifier and pClamp 6.0 software (Axon Instruments). The external solution consisted of (mM) 5 KCl, 100 NaCl, 2 MgCl₂, 10 HEPES, 0.3 CaCl₂. Niflumic acid (10 µM) was added to block the Ca²⁺-dependent Cl^– current. The external pH was adjusted to 7.4 with NaOH and the pipette was filled with 3 M KCl. Ba²⁺-containing solutions were superfused until steady-state block occurred (generally after 10 min) at each concentration before repeating full voltage-clamp protocols. Glass microelectrodes (3 M KCl-filled) had 1.3–1.8 M resistances. All cRNA transcriptions were performed with the mMESSAGE mMACHINE kit (Ambion Inc., Austin, TX, USA).

2.2 Cell isolation
Human right ventricular myocytes were obtained via endomyocardial biopsies from patients undergoing routine follow-up after heart transplantation. Ca²⁺-tolerant myocytes were isolated and stored for use within 12 h according to previously-described procedures [8]. Written informed consent was obtained as approved by the Ethics Committee of the Montreal Heart Institute. Cells were dispersed by trituration with a Pasteur pipette and stored in a high K⁺-storage medium (in mM: KCl 20, K-glutamate 70, KH₂PO₄ 10, glucose 25, β-hydroxybutyric acid 10, taurine 20, EGTA 10, albumin 0.1%, and mannitol 40) at 4°C. All chemicals were purchased from Sigma Chemical Co., St Louis, MO, USA, unless otherwise specified.

2.3 Whole-cell patch-clamp recordings
Experiments were conducted at 22°C and ionic currents were recorded under whole-cell voltage-clamp mode using an Axopatch 200B amplifier (Axon Instruments). Borosilicate glass electrodes (1 mm O.D.) had tip resistances of 2–4 M when filled with pipette solution (mM): 0.1 GTP, 110 K-aspartate, 20 KCl, 1 MgCl₂, 5 Mg–ATP, 10 HEPES, 5 EGTA, 5 Na₂-phosphocreatine. The pH was adjusted to 7.4 with NaOH. Command pulses were generated by a D–A converter controlled by pClamp 6.05 software (Axon Instruments). Tip potentials were zeroed before formation of the membrane–pipette seal in Tyrode solution. The capacitance and series resistance (Rs) were electrically compensated to minimize the duration of the capacitive surge on the current recording and the voltage drop across the clamped cell membrane. 200 µM Cd²⁺, 500 µM 4-aminopyridine (4-AP) and 1 µM atropine were added to the extracellular solution to inhibit current contamination by L-type Ca²⁺-current, transient outward K⁺-current and acetylcholine-dependent K⁺-current, respectively. Adenosine triphosphate (ATP)-dependent K⁺-current was minimized by including ATP (Mg-salt, 5 mM) in the pipette and glyburide (10 µM) in the bath solution.

2.4 Statistical analysis
Data analysis was conducted with Axon Clampfit 6, SPSS 11, Graphpad Prism 3 and Microsoft Excel 2000. Group data are expressed as the mean±S.E.M. Statistical comparisons were made with one-way ANOVA followed by Tukey's post-test (SPSS 11). A two-tailed probability P<0.05 was taken to indicate statistical significance. Concentration–response data were analyzed using curve-fitting functions of Prism 3 software (GraphPad Software Inc. CA, USA).

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
Fig. 1 shows original recordings in oocytes injected with Kir2.1 (A), Kir2.2 (B) or Kir2.3 (C) cRNA and from a human cardiomyocyte (D). Left panels show currents recorded in the absence of Ba²⁺ (Control). Left middle panels, right middle panels and right panels show currents in the presence of 10 µM, 100 µM and 1 mM Ba²⁺, respectively. Ba²⁺-block was concentration- and voltage-dependent for all currents. Fig. 2 shows respective Kir2 mean end-pulse current–voltage relationships under control conditions and in the presence of 1, 10 and 100 µM Ba²⁺. Kir2.1 and Kir2.3 showed similar Ba²⁺-sensitivity, with high-level block requiring 100 µM Ba²⁺. Both Kir2.2 and cardiac I_K1 were reduced by >60% with 10 µM Ba²⁺ and completely blocked by 100 µM Ba²⁺, indicating qualitatively-similar sensitivity that was greater than that of Kir2.1 or Kir2.3.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Original current recordings obtained from oocytes injected with Kir2.1 (A), Kir2.2 (B) or Kir2.3 (C) and from human right ventricular myocytes (D) under control conditions (left panels) and in the presence of 10 µM (left middle panels), 100 µM (right middle panels) and 1 mM (right panels) Ba2+. Kir2 currents were elicited by voltage steps from a holding potential of –60 mV to step potentials between –150 mV and +30 mV in 10 mV increments as shown by the voltage protocol in the inset. IK1 was elicited by voltage steps from a holding potential of 0 mV to step potentials between –120 mV and +40 mV in 10 mV increments as shown by the voltage protocol in the inset.

 

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Mean±S.E.M. of current–voltage relations under steady-state conditions without drug (Control, squares) and in the presence of 1, 10 and 100 µM Ba2+ (triangles, circles and diamonds, respectively). n = 14, 9, 11 and 10 cells for Kir2.1 (A), Kir2.2 (B), Kir2.3 (C) and cardiac IK1 (D).

 
Fig. 3 shows original current recordings obtained from Xenopus oocytes co-injected with both Kir2.1 and Kir2.2 (A), Kir2.1 and Kir2.3 (B) or Kir2.2 and Kir2.3 (C) cRNA, as well as current recordings from a ventricular myocyte (D), under control conditions (left panels) and in the presence of 10, 100 µM and 1 mM Ba²⁺. The response of co-injected oocytes was qualitatively like that of the human cardiomyocyte. Corresponding mean current–voltage relationships are shown in Fig. 4. The current–voltage relationships of co-injected oocytes qualitatively resemble those of cardiac I_K1 in both the absence of Ba²⁺ and in the presence of various Ba²⁺ concentrations. The Ba²⁺-sensitivity of homomeric channels and channels resulting from co-injected Kir2 subunits showed significant group differences over the entire voltage range tested (–150 to –90 mV, P<0.001).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Original current recordings obtained from oocytes co-injected with Kir2.1/Kir2.2 (A), Kir2.1/Kir2.3 (B) or Kir2.2/2.3 (C) and from a human right ventricular myocyte (D) under control conditions (left panels) and in the presence of 10 µM (left middle panels), 100 µM (right middle panels) and 1 mM (right panels) Ba2+. Kir2 currents were elicited by voltage steps from a holding potential of –60 mV to step potentials between –150 mV and +30 mV in 10 mV increments, as shown by the voltage protocol in the inset. IK1 was elicited by voltage steps from a holding potential of 0 mV to test potentials between –120 mV and +40 mV in 10 mV increments, as shown by the voltage protocol in the inset.

 

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Mean±S.E.M. of current–voltage relations under steady-state conditions without drug (control, squares) and in the presence of 1, 10 and 100 µM Ba2+ (triangles, circles and diamonds, respectively). n = 7, 6, 4 and 10 cells for Kir2.1/Kir2.2 (A), Kir2.1/Kir2.3 (B), Kir2.2/Kir2.3 (C) and cardiac IK1 (D).

 
3.1 Concentration and time dependence of Ba²⁺ block of homomeric Kir2 subunits and cardiac I_K1
To pursue the issue of Ba²⁺-block of the various constructs studied, we analyzed the block quantitatively. Fig. 5, left panels, shows original currents recorded from one oocyte before and after each of three Ba²⁺ concentrations for cells expressing Kir2.1 (A), Kir2.2 (B) or Kir2.3 (B), along with corresponding data from a human cardiomyocyte (D). For clarity, currents obtained at only one voltage (upon hyperpolarization to –120 mV) are shown. Whereas Kir2.1 and Kir2.3 showed similar Ba²⁺ sensitivity (approximately 30% current reduction at end of pulse by 10 µM Ba²⁺, Fig. 5A and C), Kir2.2 was approximately one order of magnitude more sensitive to Ba²⁺ than Kir2.1 or Kir2.3. For example, 10 µM Ba²⁺ almost completely inhibited Kir2.2 end-pulse current (Fig. 5B, left), similar to the effect of 100 µM Ba²⁺ on Kir2.1 (Fig. 5A, left) or Kir2.3 (Fig. 5C, left). Cardiac I_K1 was more sensitive to Ba²⁺ than Kir2.1 or Kir2.3 (e.g., approximately 70% end-pulse current inhibition by 10 µM Ba²⁺, Fig. 5D, left), displaying Ba²⁺-sensitivity more similar to that of Kir2.2.

View larger version (40K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Ba2+-block of currents resulting from the expression of Kir2.1 (A), Kir2.2 (B), Kir2.3 (C) and human ventricular IK1 (D). Left panels show original recordings obtained from one oocyte under control conditions and in the presence of 1, 10 and 100 µM Ba2+. Currents were elicited by steps from a holding potential of –60 mV to –120 mV in oocytes and by steps from 0 mV to –120 mV in myocytes, as shown in the protocols in the insets. Kinetics of Ba2+ block in the same oocyte or myocyte, respectively, are shown as fractional block in the middle panels. Fractional block was calculated as control current (ICtl) minus current in the presence of Ba2+ (IBa) divided by control current ([ICtl–IBa]/ICtl). The positions of the t1/2s for each example are shown by arrows. The right panels show corresponding mean±S.E.M. concentration–response curves based on end-pulse block at each concentration upon hyperpolarization to –120 mV (n = 11, 9, 10 and 10 cells for Kir2.1, Kir2.2, Kir2.3 and cardiac IK1, respectively).

 
The time-dependence of block is illustrated in the middle column of Fig. 5. Fractional block was calculated as control current (I_Ctl) minus current in the presence of Ba²⁺ (I_Ba) divided by control current ([I_Ctl–I_Ba]/I_Ctl), and expressed as a function of time during the pulse. For Kir2.1 and 2.3, block at 10 µM was virtually time-independent. At the same concentration, block of Kir2.2 showed a very rapid onset. The time-dependent block of cardiac I_K1 was substantial at 10 µM and showed a slow onset. The onset of Ba²⁺ block was quantified as the time for 50% of steady-state time-dependent block (t_1/2), since the kinetics did not consistently follow simple mono- or biexponential models. Block onset was analyzed at approximately equi-effective concentrations (10 µM for Kir2.2 and I_K1, 100 µM for Kir2.1 and 2.3). The t_1.2 values of homomeric constructs were smaller than the value for I_K1 (Table 1).

View this table: [in this window] [in a new window]   Table 1 Potency and time-course of Ba2+ block of various constructs

 
The right panels show mean concentration–response curves of the form B = 1/(1+IC₅₀/C), where B is Ba²⁺-block at any concentration C and IC₅₀ is the 50% blocking concentration. The Ba²⁺ IC₅₀s were comparable for Kir2.1 and 2.3 (16.2±3.6 and 18.5±2.1 µM respectively), about an order of magnitude greater than for Kir2.2 (2.3±0.4 µM). Cardiac I_K1 had an IC₅₀ (4.7±0.4 µM) of the same order as that of Kir2.2 and much smaller than for Kir2.1 or 2.3 (Table 1).

3.2 Concentration and time dependence of Ba²⁺-block of co-injected Kir2 subunits and cardiac I_K1
Fig. 6, left panels show original current recordings obtained from Xenopus oocytes co-injected with either Kir2.1/Kir2.2 (A), Kir2.1/Kir2.3 (B) or Kir2.2/Kir2.3 (C), in comparison with human cardiac I_K1 (D), upon hyperpolarization to –120 mV. Currents resulting from co-injected Kir2 subunits showed qualitatively similar Ba²⁺-sensitivity to cardiac I_K1. The middle column shows time-dependent block onset. Like I_K1, block of Kir2.1/Kir2.2 and Kir2.1/2.3 showed significant, slow time-dependent onset at 10 µM Ba²⁺. The t_1/2 values for Kir2.1/2.2 and Kir2.1/2.3 co-injected were similar to those of I_K1 (Table 1). The t_1/2 was somewhat faster for Kir2.2/2.3 subunits. The corresponding concentration–response relations are shown in the right column. In contrast to the homomeric subunit-based currents, the currents resulting from co-injection showed similar and low IC₅₀s (Table 1). This was the case even for Kir2.1/Kir2.3 currents, despite the fact that both Kir2.1 and Kir2.3 had much higher IC₅₀s when expressed alone. The IC₅₀s for all co-injected subunit combinations were comparable to the values for I_K1.

View larger version (40K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Ba2+ block of currents resulting from the co-expression of Kir2.1/Kir2.2 (A), Kir2.1/Kir2.3 (B), Kir2.2/Kir2.3 (C) and human ventricular IK1 (D). Left panels show original recordings obtained from one oocyte under control conditions and in the presence of 1, 10 and 100 µM Ba2+. Currents were elicited by steps from a holding potential of –60 mV to –120 mV in oocytes and by steps from 0 mV to –120 mV in myocytes, as shown in the protocol in the inset. Kinetics of Ba2+-block in the same oocyte or myocyte, respectively, are shown as fractional block in the right panels. Fractional block was calculated as control current (ICtl) minus current in the presence of Ba2+ (IBa) divided by control current ([ICtl–IBa]/ICtl). The positions of the t1/2s are indicated by arrows in the middle panels. The right panels show corresponding mean±S.E.M. concentration–response curves based on end-pulse block at each concentration upon hyperpolarization to –120 mV (n = 7, 6, 4 and 10 cells for Kir2.1, Kir2.2, Kir2.3 and cardiac IK1, respectively).

 
3.3 Comparison of Ba²⁺ blocking properties of homomeric and co-injected Kir2 subunits with cardiac I_K1
Fig. 7 shows a comparison of block at –120 mV for each pair of co-injected Kir2 subunits with that of I_K1 (middle panels) and for each of the corresponding homomeric currents with I_K1 (left panels). The right panels show Ba²⁺ IC₅₀ values as a function of voltage for each of the corresponding currents. The asterisks in the right panels show the statistical significance of differences for blocking potency at each voltage versus that of I_K1. For Kir2.1 and 2.2 (A), block is less potent for Kir2.1 and more potent for Kir2.2 at –120 mV compared to cardiac I_K1 (left panel). Block of Kir2.1/2.2 co-injected currents at –120 mV is quite similar to that of I_K1 (middle). Comparing results at all voltages (right panels), results for Kir2.1 were significantly different from those of I_K1. Results for Kir2.2, 2.1/2.3 and I_K1 were not statistically distinguishable. For Kir2.1 and 2.3 (panel B), either homomer resulted in currents less sensitive to Ba²⁺ than I_K1 (left panel), but co-injected oocytes showed a sensitivity comparable to that of I_K1 (middle panel). Examining the entire voltage range, homomeric Kir2.1 and 2.3 were each significantly less sensitive than currents resulting from Kir2.1/2.3 co-injection, which were indistinguishable from I_K1 (right). The corresponding data for Kir2.2 and 2.3 (C) indicates that once again co-injected oocytes have currents with Ba²⁺-sensitivity similar to that of I_K1.

View larger version (39K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Comparison of mean±S.E.M. concentration–response curves based on end-pulse block at each concentration at a test potential of –120 mV. Left panels compare Ba2+ block of currents carried by homomeric Kir2 channels with cardiac IK1. Heteromeric channels composed of subunits shown in left panels are compared to cardiac IK1 in the middle panels. Right panels: comparison of homomeric and heteromeric Kir2 and cardiac IK1 mean Ba2+ IC50s at test potentials between –120 mV and –90 mV. *P<0.05, **P<0.01, ***P<0.001 versus cardiac IK1 at the same voltage. (A) left panels: Kir2.1, Kir2.2 and cardiac IK1 (open squares, open circles and filled diamonds, respectively; n = 11, 9 and 10 cells for Kir2.1, Kir2.2 and cardiac IK1). Middle panels: Kir2.1/Kir2.2 (open diamonds, n = 7) and cardiac IK1 (filled diamonds; n = 10). (B) left panels: Kir2.1 (open squares, n = 11), Kir2.3 (filled circles, n = 10) and cardiac IK1 (filled diamonds, n = 10). Middle panels: Kir2.1/Kir2.3 (open diamonds, n = 6) and cardiac IK1 (filled diamonds, n = 10). (C) left panels: Kir2.2 (open circles, n = 9), Kir2.3 (filled circles, n = 10) and cardiac IK1 (filled diamonds, n = 10). Middle panels: Kir2.2/Kir2.3 (open diamonds, n = 4) and cardiac IK1 (filled diamonds, n = 10).

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
We have compared the Ba²⁺-blocking response of currents carried by homomeric channels composed of Kir2.1, Kir2.2 and Kir2.3 with the response of currents resulting from co-expressed subunits and with human cardiac I_K1. The principle novel findings are that the response of co-expressed subunit-currents is not the simple algebraic sum of individual subunit responses, and that the response of native human I_K1 is more similar to that of co-injected subunits than of each subunit expressed alone.

4.1 Importance and molecular basis of I_K1
I_K1 is crucial in cardiac electrophysiology [2,11], setting the resting potential and contributing to terminal repolarization [5]. Resting potential depolarization has complex effects on cardiac excitability (Dominguez and Fozzard, ca. 1975). Mild depolarization increases excitability by decreasing the depolarization needed to reach threshold, but stronger depolarization decreases excitability and conduction velocity by inactivating Na⁺-channels. Increased excitability facilitates arrhythmias arising from abnormal automaticity and triggered activity, whereas impaired conduction promotes re-entry. Impaired phase-3 repolarization caused by I_K1 dysfunction could lead to excess action potential prolongation and early afterdepolarizations. Thus, I_K1 abnormality can promote a wide range of arrhythmia mechanisms, any of which might be implicated in ventricular tachyarrhythmias associated with Kir2.1 dysfunction in Andersen's Syndrome [6].

Our understanding of the molecular basis of I_K1 is limited. At the mRNA level, the most abundant Kir2 subunit is Kir2.1, with ventricular concentrations that are >10-fold greater than those of Kir2.2 or Kir2.3 [8]. Although Kir2.1–4 have been detected in the heart [12–17], only Kir2.1–3 appear to be expressed in cardiomyocytes, whereas Kir2.4 is found in cardiac neuronal tissue [7]. There is 78% greater abundance of Kir2.1 protein in ventricle versus atrium and 228% greater abundance of Kir2.3 in atrium versus ventricle, but the relative quantities of Kir2.1 vs. Kir2.3 protein are unknown [18]. Unitary I_K1 conductances change with development, suggesting developmentally-based molecular alterations [19,20]. Four different I_K1 single-channel conductances are displayed in human atrial cells, compatible with molecular heterogeneity [15]. Kir2.1 anti-sense oligonucleotides suppressed, but did not eliminate, I_K1 in rat ventricular myocytes, but residual current was detected, suggesting other contributors [21]. In mice genetically engineered to lack Kir2.1 completely, I_K1 is absent in the presence of physiological (4 mM) extracellular [K⁺] [22]. Knock-out of Kir2.2 reduces I_K1 by 50% [22]. These results suggest that Kir2.1 is a component of virtually all murine I_K1 channels, whereas Kir2.2 subunits are present in about half. This possibility would be most easily explained by the formation of Kir2.1–Kir2.2 heteromers [22].

4.2 Potential role of Kir2 heteromultimers
Initial studies suggested possible co-assembly between Kir2.1 and Kir2.3 subunits, mediated by N-terminal interactions [23]. Subsequent work suggested little or no heteromultimer formation between Kir2.1 and 2.2 or 2.3, with determinants for co-assembly localized to the M2 segment and proximal C-terminus [24]. Preisig-Müller et al. recently provided extensive evidence for Kir2.1, Kir2.2 and Kir2.3 heteromultimer formation [9]. They showed that concatemers of different Kir2 subunits form functional channels, that dominant negative Kir2.1, Kir2.2 or Kir2.3 constructs suppress currents carried by wild-type subunits of each subtype upon co-injection, that Kir2.1 and Kir2.3 subunits can be co-immunoprecipitated, that cytosolic carboxy terminal domains play a key role in protein–protein interaction, and that Andersen syndrome Kir2.1 mutations have dominant-negative effects upon co-expression with Kir2.1, Kir2.2 or Kir2.3.

We have shown that Kir2.1 and 2.4 subunits co-assemble, and that Ba²⁺-blocking properties of co-assembled channels differ from homomeric Kir2.1 or 2.4 [10]. In fact, the Ba²⁺-sensitivity of Kir2.1–2.4 channels was greater than those of either Kir2.1 or 2.4 alone, suggesting that heteromeric channels are not merely an intermediate hybrid form, but may have distinct properties of their own [10]. In our study of Kir2.1–Kir2.4 interaction, as in Preisig-Müller's studies of Kir2.1, Kir2.2 and Kir2.3 interaction, currents carried by co-injected subunits behaved like channels formed by concatemers (which of necessity consist of co-assembled subunits, since the subunits are covalently linked). In the present study, currents carried by Kir2.2 co-injected with either Kir2.1 or 2.3 subunits had a Ba²⁺ IC₅₀ indistinguishable from Kir2.2-subunit homomeric channels, rather than between the IC₅₀ of Kir2.2 and the larger IC₅₀ of Kir2.1 or Kir2.3. Moreover, the IC₅₀ of co-injected Kir2.1/Kir2.3 is lower than for either homomeric subunit and of the same order as the IC₅₀ for Kir2.2. This observation resembles our previous findings for Kir2.1/Kir2.4 and supports the notion of distinct properties for heteromeric Kir2 channels.

The present study is the first direct comparison between properties of currents carried by combinations of Kir2 subunits with those of native I_K1. The response to Ba²⁺ of currents carried by all combinations of co-injected subunits was more similar to that of human I_K1 in terms of sensitivity, and Kir2.1/2.2 and Kir2.1/2.3 current Ba²⁺ blocking kinetics were more like those of I_K1, than homomeric channels. This finding supports the notion that a significant portion of I_K1 may be carried by Kir2 heteromers, as initially suggested by Zaritsky et al. [22] and recently reinforced by the elegant studies of Preisig-Müller and co-workers [9].

Our results help to resolve an issue arising from a recent study that compared homomeric guinea-pig Kir2 subunits to guinea-pig cardiac I_K1 [7]. Based upon Ba²⁺-blocking properties, the authors concluded that Kir2.2 was likely the predominant subunit underlying I_K1. However, in both human [8] and mouse [22] heart, Kir2.1 transcripts are much more abundant than those encoding Kir2.2. We found that Ba²⁺-blocking sensitivity of heteromeric Kir2 channels is similar to those of Kir2.2. Thus, the guinea-pig findings [7] are compatible with a prominent role for heteromeric Kir2 channels in native I_K1, and do not necessarily imply predominance of Kir2.2 per se. This notion would fit well with results of Kir2.1 knock-down [21] and knock-out [22] studies, while being agreeing with the guinea-pig Ba²⁺-sensitivity data [7].

4.3 Potential limitations
We did not study single-channel Kir2 or I_K1 properties. Single-channel studies of native I_K1 have provided a wide variety of results. Wible et al. described four distinct conductances (41, 35, 21 and 9 pS) in human atrium [15], whereas Liu et al. found only three conductances (10.5, 22 and 32.5 pS) in guinea-pig cardiomyocytes [7]. Nakamura et al. described six diverse conductances (8, 14, 21, 35, 43 and 80 pS) in rat ventricle [21]. Conductances of native I_K1 channels may differ from those of heterologously-expressed Kir2 subunits of the same species [7]. Furthermore, homomeric mouse Kir2.1 channels show a broad range of conductances ranging from 2 to 33 pS [25]. Thus, single-channel analyses have not to date provided clear insights into the molecular composition of I_K1 channels.

	5. Conclusions

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
This is the first study to compare properties of heteromeric Kir2 currents with those of native cardiac I_K1. Our results support the notion that a substantial proportion of native I_K1 may result from heteromultimer formation among Kir2 subunits.

Time for primary review 35 days.

	Acknowledgements

 
The authors thank Chantal St-Cyr, Evelyn Landry, and Xiao Fan Yang for excellent technical assistance, and Daniela Giuliani, France Thériault and Diane Campeau for secretarial help with the manuscript. They thank Lily Jan for providing Kir2.1, Barbara Wible for Kir2.2, and Carol Vandenberg for Kir2.3. Dr Schram was supported by the Ernst and Berta Grimmke Foundation, the Canadian Institutes of Health Research (CIHR) and Aventis Pharma. Marc Pourrier was supported by a Heart and Stroke Foundation of Canada studentship. Zhiguo Wang and Michel White are research scholars of the Fonds de la recherche en santé du Québec. Operating support was received from CIHR, the Quebec Heart and Stroke Foundation, and the Mathematics of Information Technology And Complex Systems (MITACS) Network.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 

1. Katz B. Les constantes électrique de la membrane du muscle. Arch Sci Physiol (1949) 3:285–299.
2. Nichols C.G., Lopatin A.N. Inward rectifier potassium channels. Annu Rev Physiol (1997) 59:171–191.[CrossRef][Web of Science][Medline]
3. Sakmann B., Trube G. Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart. J Physiol (Lond) (1984) 347:641–657.[Abstract/Free Full Text]
4. Ibarra J., Morley G.E., Delmar M. Dynamics of the inward rectifier K⁺ current during the action potential of guinea pig ventricular myocytes. Biophys J (1991) 60:1534–1539.[Web of Science][Medline]
5. Shimoni Y., Clark R.B., Giles W.R. Role of an inwardly rectifying potassium current in rabbit ventricular action potential. J Physiol (Lond) (1992) 448:709–727.[Abstract/Free Full Text]
6. Plaster N.M., Tawil R., Tristani-Firouzi M., et al. Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. Cell (2001) 105:511–519.[CrossRef][Web of Science][Medline]
7. Liu G.X., Derst C., Schlichthorl G., et al. Comparison of cloned Kir2 channels with native inward rectifier K⁺ channels from guinea-pig cardiomyocytes. J Physiol (Lond) (2001) 532:115–126.[Abstract/Free Full Text]
8. Wang Z., Yue L., White M., Pelletier G., Nattel S. Differential distribution of inward rectifier potassium channel transcripts in human atrium versus ventricle. Circulation (1998) 98:2422–2428.[Abstract/Free Full Text]
9. Preisig-Muller R., Schlichthorl G., George T., et al. Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome. Proc Natl Acad Sci USA (2002) 99:7774–7779.[Abstract/Free Full Text]
10. Schram G., Melnyk P., Pourrier M., Wang Z., Nattel S. Kir2.4 and Kir2.1 K⁺ channel subunits co-assemble: a potential new contributor to inward rectifier current heterogeneity. J Physiol (Lond) (2002) 544(2):337–349.[Abstract/Free Full Text]
11. Lopatin A.N., Nichols C.G. Inward rectifiers in the heart: an update on I(K1). J Mol Cell Cardiol (2001) 33:625–638.[CrossRef][Web of Science][Medline]
12. Ishii K., Yamagishi T., Taira N. Cloning and functional expression of a cardiac inward rectifier K⁺ channel. FEBS Lett (1994) 338:107–111.[CrossRef][Web of Science][Medline]
13. Raab-Graham K.F., Radeke C.M., Vandenberg C.A. Molecular cloning and expression of a human heart inward rectifier potassium channel. NeuroReport (1994) 5:2501–2555.[Web of Science][Medline]
14. Ashen M.D., O'Rourke B., Kluge K.A., Johns D.C., Tomaselli G.F. Inward rectifier K⁺ channel from human heart and brain: cloning and stable expression in a human cell line. Am J Physiol (Heart Circ Physiol) (1995) 268:H506–H511.[Abstract/Free Full Text]
15. Wible B.A., De Biasi M., Majumder K., Taglialatela M., Brown A.M. Cloning and functional expression of an inwardly rectifying K⁺ channel from human atrium. Circ Res (1995) 76:343–500.[Abstract/Free Full Text]
16. Wood L.S., Tsai T.D., Lee K.S., Vogeli G. Cloning and functional expression of a human gene, hIRK1, encoding the heart inward rectifier K⁺-channel. Gene (1995) 163:313–317.[CrossRef][Web of Science][Medline]
17. Topert C., Doring F., Wischmeyer E., et al. Kir2.4: a novel K⁺ inward rectifier channel associated with motoneurons of cranial nerve nuclei. J Neurosci (1998) 18:4096–4105.[Abstract/Free Full Text]
18. Melnyk P., Zhang L., Shrier A., Nattel S. Differential distribution of Kir2.1 and Kir2.3 subunits in canine atrium and ventricle. Am J Physiol (Heart Circ Physiol) (2002) 283:H1123–H1133.[Abstract/Free Full Text]
19. Josephson I.R., Sperelakis N. Developmental increases in the inwardly-rectifying K⁺ current of embryonic chick ventricular myocytes. Biochim Biophys Acta (1990) 1052:123–127.[Medline]
20. Wahler G.M. Developmental increases in the inwardly rectifying potassium current of rat ventricular myocytes. Am J Physiol (1992) 262:C1266–C1272.[Web of Science][Medline]
21. Nakamura T.Y., Artman M., Rudy B., Coetzee W.A. Inhibition of rat ventricular I_K1 with antisense oligonucleotides targeted to Kir2.1 mRNA. Am J Physiol (1998) 274:H892–H900.[Web of Science][Medline]
22. Zaritsky J.J., Redell J.B., Tempel B.L., Schwarz T.L. The consequences of disrupting cardiac inwardly rectifying K(+) current (I(K1)) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes. J Physiol (Lond) (2001) 533:697–710.[Abstract/Free Full Text]
23. Fink M., Duprat F., Heurteaux C., et al. Dominant negative chimeras provide evidence for homo and heteromultimeric assembly of inward rectifier K⁺ channel proteins via their N-terminal end. FEBS Lett (1996) 378:64–68.[CrossRef][Web of Science][Medline]
24. Tinker A., Jan Y.N., Jan L.Y. Regions responsible for the assembly of inwardly rectifying potassium channels. Cell (1996) 87:857–868.[CrossRef][Web of Science][Medline]
25. Picones A., Keung E., Timpe L.C. Unitary conductance variation in Kir2.1 and in cardiac inward rectifier potassium channels. Biophys J (2001) 81:2035–2049.[Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				H.-A. Lee, K.-S. Kim, S.-J. Park, and E.-J. Kim Cellular Mechanism of the QT Prolongation Induced by Sulpiride International Journal of Toxicology, May 1, 2009; 28(3): 207 - 212. [Abstract] [Full Text] [PDF]

				M. Maeda, E. Tanaka, K. Shoudai, K. Nonaka, N. Murayama, Y. Ito, and N. Akaike Differential Effects of Divalent Cations on Spontaneous and Evoked Glycine Release From Spinal Interneurons J Neurophysiol, February 1, 2009; 101(2): 1103 - 1113. [Abstract] [Full Text] [PDF]

				P. D. Smith, S. E. Brett, K. D. Luykenaar, S. L. Sandow, S. P. Marrelli, E. J. Vigmond, and D. G. Welsh KIR channels function as electrical amplifiers in rat vascular smooth muscle J. Physiol., February 15, 2008; 586(4): 1147 - 1160. [Abstract] [Full Text] [PDF]

				B. K. Panama, M. McLerie, and A. N. Lopatin Heterogeneity of IK1 in the mouse heart Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3558 - H3567. [Abstract] [Full Text] [PDF]

				M. R. Kasten, B. Rudy, and M. P. Anderson Differential regulation of action potential firing in adult murine thalamocortical neurons by Kv3.2, Kv1, and SK potassium and N-type calcium channels J. Physiol., October 15, 2007; 584(2): 565 - 582. [Abstract] [Full Text] [PDF]

				M. Hassinen, V. Paajanen, J. Haverinen, H. Eronen, and M. Vornanen Cloning and expression of cardiac Kir2.1 and Kir2.2 channels in thermally acclimated rainbow trout Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2007; 292(6): R2328 - R2339. [Abstract] [Full Text] [PDF]

				M. C. Jantzi, S. E. Brett, W. F. Jackson, R. Corteling, E. J. Vigmond, and D. G. Welsh Inward rectifying potassium channels facilitate cell-to-cell communication in hamster retractor muscle feed arteries Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1319 - H1328. [Abstract] [Full Text] [PDF]

				B. K. Panama and A. N. Lopatin Differential polyamine sensitivity in inwardly rectifying Kir2 potassium channels J. Physiol., March 1, 2006; 571(2): 287 - 302. [Abstract] [Full Text] [PDF]

				Y. Fang, G. Schram, V. G. Romanenko, C. Shi, L. Conti, C. A. Vandenberg, P. F. Davies, S. Nattel, and I. Levitan Functional expression of Kir2.x in human aortic endothelial cells: the dominant role of Kir2.2 Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1134 - C1144. [Abstract] [Full Text] [PDF]

				D.-H. Yan, K. Nishimura, K. Yoshida, K. Nakahira, T. Ehara, K. Igarashi, and K. Ishihara Different intracellular polyamine concentrations underlie the difference in the inward rectifier K+ currents in atria and ventricles of the guinea-pig heart J. Physiol., March 15, 2005; 563(3): 713 - 724. [Abstract] [Full Text] [PDF]

				K. Goto, N. M Rummery, T. H. Grayson, and C. E Hill Attenuation of conducted vasodilatation in rat mesenteric arteries during hypertension: role of inwardly rectifying potassium channels J. Physiol., November 15, 2004; 561(1): 215 - 231. [Abstract] [Full Text] [PDF]

				E. Zitron, C. Kiesecker, S. Luck, S. Kathofer, D. Thomas, V. A.W Kreye, J. Kiehn, H. A Katus, W. Schoels, and C. A Karle Human cardiac inwardly rectifying current IKir2.2 is upregulated by activation of protein kinase A Cardiovasc Res, August 15, 2004; 63(3): 520 - 527. [Abstract] [Full Text] [PDF]

				A. S. Dhamoon, S. V. Pandit, F. Sarmast, K. R. Parisian, P. Guha, Y. Li, S. Bagwe, S. M. Taffet, and J. M.B. Anumonwo Unique Kir2.x Properties Determine Regional and Species Differences in the Cardiac Inward Rectifier K+ Current Circ. Res., May 28, 2004; 94(10): 1332 - 1339. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Schram, G. Articles by Nattel, S. Search for Related Content PubMed PubMed Citation Articles by Schram, G. Articles by Nattel, S. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics