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J. Biol. Chem., Vol. 277, Issue 39, 35815-35818, September 27, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/39/35815    most recent M206032200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Collins, A. Articles by Larson, M. Search for Related Content PubMed PubMed Citation Articles by Collins, A. Articles by Larson, M. Social Bookmarking                      What's this?

Differential Sensitivity of Inward Rectifier K⁺ Channels to Metabolic Inhibitors^*

Anthony Collins and Maureen Larson

From the Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331-3507

Received for publication, June 18, 2002, and in revised form, July 8, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibition of inward rectifier K⁺ channels under ischemic conditions may contribute to electrophysiological consequences of ischemia such as cardiac arrhythmia. Ischemia causes metabolic inhibition, and the use of metabolic inhibitors is one experimental method of simulating ischemia. The effects of metabolic inhibitors on the activity of inward rectifier K⁺ channels K_ir2.1, K_ir2.2, and K_ir2.3 were studied by heterologous expression in Xenopus oocytes and two-electrode voltage clamp. 10 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited K_ir2.2 and K_ir2.3 currents but was without effect on K_ir2.1 currents. The rate of decline of current in FCCP was faster for K_ir2.3 than for K_ir2.2. K_ir2.3 was inhibited by 3 mM sodium azide (NaN₃), whereas K_ir2.1 and K_ir2.2 were not. K_ir2.2 was inhibited by 10 mM NaN₃. All three of these inward rectifiers were inhibited by lowering the pH of the solution perfusing inside-out membrane patches. K_ir2.3 was most sensitive to pH (pK = 6.9), whereas K_ir2.1 was least sensitive (pK = 5.9). For K_ir2.2 the pK was 6.2. These results demonstrate the differential sensitivity of these inward rectifiers to metabolic inhibition and internal pH. The electrophysiological response of a particular cell type to ischemia may depend on the relative expression levels of different inward rectifier genes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inward rectifier K⁺ channels are known to be involved in the electrophysiology of several different tissues, including the heart (1). In fast response myocardial tissue they are responsible for the relatively negative value of the resting membrane potential and the late rapid repolarization phase of the action potential (2). Their characteristic property of inward rectification allows the myocardial cell to generate long lasting action potentials while minimizing ion fluxes (3).

Ischemia produces electrophysiological changes that can lead to the generation of cardiac arrhythmias (4) or cerebral excitotoxicity (5). Simulated ischemia has been shown to inhibit an inwardly rectifying current in isolated cardiac myocytes (6). This effect may contribute to arrhythmogenesis and is therefore worthy of further study. Three closely related inward rectifier genes, K_ir2.1, K_ir2.2, and K_ir2.3, are expressed in myocardium (7, 8), so each of these could potentially be involved in the aforementioned ischemic response in the heart. These genes are also expressed in the brain (9-11). In this report we have simulated ischemia by applying metabolic inhibitors to Xenopus oocytes expressing these three inward rectifier K⁺ channels. We show that K_ir2.1, K_ir2.2, and K_ir2.3 are affected differently by metabolic inhibition. We also show that the sensitivity of K_ir2.2 to internal pH is intermediate between that of K_ir2.1 and K_ir2.3.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Subcloning and in Vitro Transcription-- Complementary DNAs encoding K_ir2.1 (IRK1) (9), K_ir2.2 (MB-IRK2) (11), and K_ir2.3 (MB-IRK3) (10) were subcloned into the Xenopus expression vector pGEMHE (12). Plasmids were linearized with NheI, and cRNA was transcribed in vitro with T7 RNA polymerase (mMessage mMachine, Ambion, Austin, TX). RNA yield and integrity were assessed by agarose-ethidium bromide gel electrophoresis.

Oocyte Isolation-- Stage V-VI oocytes were surgically removed from Xenopus laevis frogs (Nasco) under anesthesia (0.03% benzocaine for 10-15 min) and incubated with 1.3 mg/ml collagenase (Worthington, type CLS3) for 2 h at 22 °C in 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES, pH 7.4, with agitation to remove connective tissue. Oocytes were then washed several times with 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.4, and stored in the same solution at 18 °C. Both solutions also contained 100 µg/ml streptomycin and 60 µg/ml ampicillin. Between 16 and 24 h later, oocytes were injected (Nanoliter; World Precision Instruments) with amounts of RNA that gave approximately equal expression levels (0.2 ng of K_ir2.1, 4 ng of K_ir2.2, and 4 ng of K_ir2.3) in 50 nl of nuclease-free water.

Two-electrode Voltage Clamp-- Inward rectifier currents were recorded 1-2 days after injection using a TEC-03 amplifier (npi, Germany) controlled by Pulse 8.4 software (Heka, Germany) via an ITC-16 computer interface (Instrutech, Long Island, NY). Currents were filtered at 500 Hz and digitized at 1 kHz. Data were analyzed using Pulse 8.4 and Prism 3.02 (GraphPad). Microelectrode pipettes had resistances of 0.5-1.5 megohms. Oocytes were placed in a 100-µl volume recording chamber that was continuously perfused at a rate of ~1.2 ml/min. Different solutions entered the chamber via a common outlet with a dead space of ~50 µl. Under control conditions oocytes were perfused with "90K" solution: 90 mM KCl/KOH, 3 mM MgCl₂, 5 mM HEPES, pH 7.4. Sodium azide (NaN₃) and BaCl₂ were dissolved in aliquots of this solution as solid. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)¹ (ICN, Costa Mesa, CA) was dissolved in dimethyl sulfoxide (Me₂SO) to 100 mM. This stock solution was diluted into 90K.

Patch Clamp Recording-- Inward rectifier currents were recorded in "giant" inside-out patches from Xenopus oocytes (13) 3-9 days after injection with K_ir2.x RNA. The patch clamp amplifier was an Axopatch 200B (Axon Instruments). Currents were filtered at 1 kHz, and data were acquired at 4.76 kHz with a Digidata 1320A computer interface and pClamp 8 software (Axon Instruments). Data were analyzed using pClamp 8 and Prism 3.02 (GraphPad). Patch pipettes had inner tip diameters of 20-25 µm and contained a "FVPP" type solution (14) (in mM): 40 KCl, 75 potassium gluconate, 5 KF, 0.1 mM NaVO₃, 10 sodium pyrophosphate, 1 mM EGTA, 10 glucose, 0.1 spermine, 10 PIPES, pH 7.4, with HCl. The recording chamber contained this same solution. Inside-out patches were perfused with this solution and with aliquots of this solution that had been further adjusted to different pH values with HCl. Switching between different perfusion solutions was achieved with a BPS-8 valve control system and a QMM micromanifold with a dead space of ~1 µl (ALA Scientific Instruments, Westbury, NY). The perfusion rate was 85 µl/min.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of FCCP on Inward Rectifier Currents in Intact Oocytes-- Inward rectifier currents were monitored by means of a continuous two-electrode voltage clamp protocol. Membrane potential was held at 0 mV and at 20-s intervals was stepped to 50 mV for 75 ms and then to 50 mV for 350 ms. Expression of inward rectifier channels was confirmed by brief application of 20 mM Ba²⁺, which completely blocks K_ir2.1, K_ir2.2, and K_ir2.3 (9-11). Oocytes were first perfused with 90K to ensure a steady current, and then the perfusion solution was switched to 90K plus 10 µM FCCP, which inhibits ATP production by dissipating the H⁺ gradient across the inner mitochondrial membrane (15). (This solution also contained 0.01% Me₂SO. Me₂SO alone had no effect on inward rectifier currents at this concentration.)

In oocytes expressing K_ir2.1, inward rectifier current remained steady in the presence of 10 µM FCCP. In contrast, 10 µM FCCP inhibited inward rectifier current in oocytes expressing K_ir2.2 or K_ir2.3. Examples of these experiments are shown in Fig. 1. To rule out the unlikely possibility that the decline of K_ir2.2 and K_ir2.3 currents was because of spontaneous "rundown," we perfused oocytes expressing these clones with 90K (plus 0.01% Me₂SO) for prolonged periods without switching to FCCP. As shown by the examples in Fig. 2, spontaneous rundown did not occur. Indeed, in some cases (as in Fig. 2) the current had a tendency to slowly increase. This was not because of an increase in leak current, as demonstrated by applying 20 mM Ba²⁺.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Examples of FCCP application to Xenopus oocytes expressing Kir2.1, Kir2.2, or Kir2.3. FCCP (10 µM) was applied in 90K solution following 1 min, 40 s of perfusion with 90K alone (plus 0.01% Me2SO). Inward rectifier current was monitored by two-electrode voltage clamp. At 20-s intervals the membrane potential was stepped from the holding potential of 0 mV to 50 mV for 75 ms and then to 50 mV for 350 ms. The data in panel A represent the current recorded at the end of the 50 mV step. After 15 min of FCCP application the oocytes were briefly exposed to 20 mM Ba2+, which was added to the 90K solution as BaCl2. Panels B, C, and D show current traces obtained at the times indicated by the letters a, b, and c, for Kir2.1, Kir2.2, and Kir2.3, respectively. The horizontal line indicates zero current level.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Examples of prolonged recordings of Kir2.2 and Kir2.3 currents in the continued presence of 90K (plus 0.01% Me2SO). The voltage clamp protocol was the same as in Fig. 1. The data in panel A represent the current recorded at 50 mV. The oocytes were briefly exposed to 20 mM Ba2+ at the times indicated. Panels B and C show current traces obtained at the times indicated by the letters a, b, and c, for Kir2.2 and Kir2.3, respectively. The horizontal line indicates zero current level.

Because the rate of decrease of inward rectifier currents in FCCP was quite slow, we chose an arbitrary time point of 15 min to compare the response of K_ir2.1, K_ir2.2, and K_ir2.3 to FCCP. Fig. 3 shows that there was a significant difference between K_ir2.2 and K_ir2.3 in terms of the fractional current remaining after 15 min of exposure to FCCP, demonstrating that the rate of K_ir2.2 current decline was slower than that of K_ir2.3. The fractional K_ir2.2 current remaining after 15 min was significantly different from that of K_ir2.1, which was unaffected by FCCP. In excised membrane patches, 10 µM FCCP was without effect on K_ir2.2 and K_ir2.3 (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Comparison of the effects of FCCP on Kir2.1, Kir2.2, and Kir2.3. Currents were recorded by two-electrode voltage clamp as in Figs. 1 and 2. The current recorded at 50 mV was used for this analysis. Oocytes were perfused with 90K plus 10 µM FCCP or 90K alone (plus 0.01% Me2SO) for 15 min. The perfusate was then switched to 90K plus 20 mM BaCl2. The current remaining in the presence of Ba2+ was subtracted from currents recorded at the beginning and end of the 15-min interval. The Ba2+-sensitive current at the end of the 15-min interval was normalized to the Ba2+-sensitive current at the beginning of the 15-min interval. A standard t test was used to compare pairs of data groups. p values are as follows: p < 0.005 for column 1 versus column 2, column 2 versus column 3, and column 2 versus column 4; p < 0.0001 for column 3 versus column 5. Bars represent means ± S.E. from 6 oocytes.

Effects of Sodium Azide on Inward Rectifier Currents in Intact Oocytes-- To gain further evidence that the effect of FCCP was because of metabolic inhibition, we tested another metabolic inhibitor, sodium azide (NaN₃) (16, 17). Voltage clamp experiments were carried out using the same protocol as that used for the FCCP experiments. As shown in Fig. 4, K_ir2.3 currents decreased significantly during 15 min of exposure to 3 mM NaN₃, whereas K_ir2.1 and K_ir2.2 currents were unaffected by this treatment. However, K_ir2.2 currents were inhibited by 10 mM NaN₃.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Comparison of the effects of NaN3 on Kir2.1, Kir2.2, and Kir2.3. The protocol was the same as for Fig. 3. A standard t test was used to compare pairs of data groups. p values are as follows: p < 0.0001 for column 1 versus column 5 and for column 4 versus column 5; p < 0.05 for column 2 versus column 3. Bars represent means ± S.E. from 6 oocytes.

Effects of Internal pH on Inward Rectifier Currents in Inside-out Patches-- It has been reported that metabolic inhibitors decrease intracellular pH (18, 19). It has also been reported that K_ir2.3 is more sensitive than K_ir2.1 to decreased intracellular pH (14). Therefore the sensitivity of K_ir2.3 to metabolic inhibitors compared with K_ir2.1 is consistent with a decrease in intracellular pH caused by metabolic inhibition. To investigate whether the intermediate sensitivity of K_ir2.2 to metabolic inhibitors could be explained in terms of pH sensitivity, we compared the dose response of the three inward rectifiers to internal pH in inside-out membrane patches. As shown in Fig. 5, the sensitivity of K_ir2.2 to decreasing pH was intermediate between that of K_ir2.1 and K_ir2.3.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   The effect of internal pH on Kir2.1, Kir2.2, and Kir2.3. A-C, examples of currents recorded in inside-out patches perfused with solutions of different pH as indicated. Membrane potential was held at 0 mV and stepped to 50 mV for 63 ms and then to 50 mV for 126 ms. The horizontal line indicates zero current level. D, pH dependence of current at 50 mV. Data are from 6 oocytes each. Data points and error bars represent the mean ± S.E. Currents were normalized to the current in pH 7.4. Curves are the best fits of the data to I/IpH7.4 = 1/(1 + ([H+]/K)n), where K is the [H+] at which the current is 50% inhibited and n is the Hill coefficient. pK values are 5.9, 6.2, and 6.9 for Kir2.1, Kir2.2, and Kir2.3, respectively. Hill coefficients are 2.2, 2.8, and 3.0 for Kir2.1, Kir2.2, and Kir2.3, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acute ischemia produces many changes in the intracellular environment. These changes affect the activity of several different ion channels, leading to cellular electrophysiological alterations that can produce arrhythmias in the heart (4) or excitotoxicity in the brain (5). Inward rectifier K⁺ channels play an important role in the electrophysiology of the cardiac myocyte (2, 20), and inhibition of their activity will have a significant and possibly arrhythmogenic effect (4). Acute ischemia produces a shortening of the action potential because of activation of ATP-sensitive K⁺ channels preceded in some cases by a lengthening. The lengthening has been attributed to inhibition of the transient outward current accompanied by inhibition of the inward rectifier current in some preparations (6) but not others (21). Further studies are required to determine the contribution of inward rectifier channels to changes in action potential duration during ischemia relative to other channel types.

The results presented here suggest that the effect of ischemia on inward rectifier current will depend on the molecular identity of the channels. We have demonstrated that the inward rectifiers K_ir2.1, K_ir2.2, and K_ir2.3 are differentially sensitive to inhibition of cellular metabolism. Because cellular metabolism is inhibited in ischemia (4), our experimental conditions simulate some of the conditions prevailing during ischemia. Therefore it seems reasonable to propose that of these three related inward rectifiers K_ir2.3 will be inhibited the most under ischemic conditions, whereas K_ir2.1 will be inhibited the least.

One of the intracellular changes that occurs during ischemia and metabolic inhibition is a decrease in pH (4, 18, 19). In this study, the order of sensitivity of K_ir2.1, K_ir2.2, and K_ir2.3 to metabolic inhibition was the same as the order of sensitivity to lowering internal pH. However, the relative sensitivity to metabolic inhibition cannot be accounted for by a change in internal pH alone. For example, the degree of inhibition of K_ir2.2 after 15 min of exposure to 10 µM FCCP (15%; Fig. 3, column 2) corresponds to an internal pH of 6.5 (Fig. 5D), whereas the degree of inhibition of K_ir2.3 after 15 min of exposure to 10 µM FCCP (38%; Fig. 3, column 3) corresponds to an internal pH of 7.0 (Fig. 5D). Therefore our results suggest the influence of other intracellular consequences of metabolic inhibition. For example, metabolic inhibitors have been shown to deplete intracellular ATP in Xenopus oocytes (22).

Experimental data and simulations indicate that a change of a few nanosiemens in the K⁺ conductance of a cardiac myocyte can significantly affect action potential duration (23). Therefore, inhibition of only a fraction of the 150 nanosiemens inward rectifier conductance in the cardiac myocyte (24) may have a significant effect in ischemia. In the heart the inward rectifier current (I_K1) is probably because of expression of both K_ir2.1 and K_ir2.2 with a small or no contribution from K_ir2.3, depending on the species (7-11, 25). However, the relative functional expression levels of these different channels are not known accurately. Inward rectifier mRNA levels have been measured in human heart, but they may not reflect expression at the functional level (8). Differences in the relative expression levels of these genes in different anatomical locations in the heart may produce spatial disparities in the refractory period in response to ischemia. Such situations are proarrhythmic (4). Details of the spatial distributions of inward rectifier gene expression in the heart are unknown at present. As an added complication, it is possible that K_ir2.2 exists as a heteromultimer with K_ir2.1 (25). Although K_ir2.3 may not contribute much to I_K1 in the heart, its location in neurons (26, 27) raises the possibility of a role in ischemia-induced excitotoxicity.

	ACKNOWLEDGEMENT

We thank Dr. Lily Jan for K_ir2.1, K_ir2.2, and K_ir2.3 clones.

	FOOTNOTES

^* This work was funded in part by the American Heart Association, Northwest Affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, 15th and Jefferson, Corvallis, OR 97331-3507. Tel.: 541-737-5799; Fax: 541-737-3999; E-mail: tony.collins@orst.edu.

Published, JBC Papers in Press, July 12, 2002, DOI 10.1074/jbc.M206032200

	ABBREVIATIONS

The abbreviations used are: FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; PIPES, 1,4-piperazinediethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/39/35815    most recent M206032200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Collins, A. Articles by Larson, M. Search for Related Content PubMed PubMed Citation Articles by Collins, A. Articles by Larson, M. Social Bookmarking                      What's this?