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Physiol. Genomics 25: 105-115, 2006. First published January 10, 2006; doi:10.1152/physiolgenomics.00106.2005
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Received 3 May 2005; accepted in final form 22 December 2005.
Physiological Genomics 25:105-115 (2006)
American Physiological Society © 2006 American Physiological Society

Elimination of allosteric modulation of myocardial KATP channels by ATP and protons in two Kir6.2 polymorphisms found in sudden cardiac death

Ningren Cui*, Li Li*, Xueren Wang*, Yun Shi, Weiwei Shi and Chun Jiang

Department of Biology, Georgia State University, Atlanta, Georgia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The major cause of sudden cardiac death (SCD) is ventricular arrhythmias due to unstable myocardial electrical activity in which the ATP-sensitive K+ (KATP) channels play a role. Genetic disruption of these channels predisposes the myocardium to arrhythmias. Two point mutations in the Kir6.2 subunit are found in SCD with acute myocardial infarction. Here we show evidence for the functional consequences of the P266T and R371H variants. Baseline single-channel properties, expression density, and channel modulations were studied in patch clamp. We focused on channel modulations by intracellular ATP and protons, as the concentration of these two important KATP channel regulators changes widely with hypoxic ischemia. We found that both variants expressed functional currents even though they occur at two highly conserved regions. The open state probability of P266T was twice as high as the wild-type (WT) channel, whereas its channel density was only ~20% of the WT channel. Although the outward current was not affected by these two mutations at neutral pH, it was ~20% lower at acidic pH in the P266T than in the WT channel. Both P266T and R371H mutations significantly reduced ATP sensitivity and increased pH sensitivity. More dramatically, allosteric regulation by intracellular ATP and protons was almost completely eliminated in the polymorphic P266T and R371H channels. Such an abnormality was seen in both inward and outward currents. Given the importance and beneficial effects of allosteric regulation in cellular responses to metabolic stress, the loss of such a regulatory mechanism in the P266T and R371H variants appears consistent with the adverse consequences occurring during acute myocardial infarction in patients.

ATP-sensitive potassium channel; arrhythmia; pH; genetic variation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
SUDDEN CARDIAC DEATH (SCD) is a leading public health problem, claiming 300,000–400,000 lives a year in the United States (42). It is defined as death of natural, nonviolent, and unexpected causes occurring within 1 h of the onset of acute symptoms (35). The World Health Organization defines sudden death as death within 24 h after onset of symptoms (9). The prognosis of SCD depends on the identification of major risk factors. In patients with structural heart disease, left ventricular dysfunction is the strongest predictor of SCD. Ventricular fibrillation and other ventricular tachyarrhythmias account for 80% of SCD (36). Common risk indexes such as plasma lipid levels, hypertension, diabetes, and smoking history, however, are not always helpful in predicting SCD (1). Recent progress in human genomics provides another approach to the identification of risk factors, as the genetic background is likely to contribute to the consequences of ischemia-induced ventricular tachycardia and thus SCD (1).

Two point mutations in the Kcnj11 gene have been identified in patients who died of SCD after myocardial infarction (14). These two polymorphisms (R371H, P266T) occur in two highly conserved regions of the pore-lining Kir6.2 subunit in the ATP-sensitive K+ (KATP) channels that play an important role in myocardium excitability. It is possible that such variations lead to dysfunction of KATP channels and affect cardiac responses to metabolic stress such as hypoxic ischemia, although these K+ channels are quiescent under physiological conditions.

The KATP channels, composed of four pore-forming Kir6.2 subunits and four sulfonylurea receptor subunits (SUR2A), are expressed in myocardium (12, 13). The sarcolemmal KATP channels regulate membrane potential and action potential duration (11, 28), whereas the mitochondrial KATP channels play an important role in ischemic preconditioning, i.e., a brief period of ischemia lessens the amount of myocardial damage produced by a subsequent prolonged ischemia (15, 32). Pharmacological KATP channel openers and blockers can change the myocardial action potential duration by affecting phase 3 repolarization (5, 25, 40). Genetic disruption of KATP channels produced defective cardiac action potential shortening, predisposing the myocardium to early afterdepolarizations and arrhythmias on catecholamine challenge (19). The myocardium from Kir6.2-knockout mice also shows survival disadvantages, as impaired cardiac performance and pathological Ca2+-dependent structural damage occur in the heart with repetitive exercises (16), suggesting that abnormal activity of the myocardial KATP channels may underscore myocardium vulnerability to arrhythmias and hypoxic ischemia.

The KATP channels are regulated by several metabolites including ATP, ADP, protons, phospholipids, and long-chain fatty acyl-CoA (7, 21, 26, 30, 38). The channel regulation by intracellular ATP and protons is remarkable. Whereas the channels are directly activated by decreases in intracellular pH (pHi) and ATP concentration, the allosteric interaction of these KATP channel regulators allows vast channel activation with moderate changes in their concentrations (34). Such an allosteric mechanism may play a more important role in myocardial KATP channel regulation, as a combined decrease in pHi and ATP is more frequently seen in muscle tissues than ATP reduction alone. To elucidate baseline properties of the P266T and R371H variants in myocardial KATP channels and their surface expression, sensitivity to intracellular ATP and protons, and effects on allosteric modulation by these two KATP channel regulators, we performed these studies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Kir6.2 (GenBank no. D50581) and SUR2A (GenBank no. D83598) cDNAs were cloned in a eukaryotic expression vector and expressed in a mammalian cell line. Site-specific mutations were produced with a site-directed mutagenesis kit based on the Pfu DNA polymerase (Stratagene, La Jolla, CA). Correct mutations were confirmed with DNA sequencing.

Human embryonic kidney cells (HEK293; CRL-1573, batch no. 2187595, American Type Culture Collection, Rockville, MD) were chosen to express the KATP channels, as these cells possess mammalian expression machineries and show little inward rectifying K+ current (41). Cells were cultured as monolayers in MEM-E medium with 10% fetal bovine serum added. The cells were cultured at 37°C with 5% CO2 and split every 3–4 days. The cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with 1 µg of wild-type (WT) or mutant Kir6.2 cDNA plus 4 µg of SUR2A cDNA per petri dish (35 mm). For heteromeric expression, 0.5 µg of WT and 0.5 µg of mutant cDNA were cotransfected with 4 µg SUR2A cDNA. To facilitate the identification of positively transfected cells, 0.5 µg of green fluorescent protein cDNA (pEGFP-N2, Clontech, Palo Alto, CA) was added to the cDNA mixture. Cells were dissociated from the monolayer with 0.01% trypsin 24–48 h after transfection. A few drops of the cell suspension were added to a petri dish and then incubated at 37°C for at least 2 h before experiments.

Patch-clamp experiments were performed at room temperature as described previously (37–39). In brief, fire-polished patch pipettes were made with 1.2-mm borosilicate glass capillaries. Inside-out and whole cell patches were performed with the Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Macroscopic currents in a cell-free condition were studied with recording pipettes of 0.5–1.0 M{Omega}. In whole cell patch clamp, tight seals (>1 G{Omega} before breaking into whole cell mode) were obtained in the transfected cells. The series resistance during recording varied from 5 to 10 M{Omega} and was not compensated. All current records were low-pass filtered (2 kHz, Bessel 4-pole filter, –3 dB), digitized (10 kHz, 16-bit resolution), and stored on computer disk for later analysis with pCLAMP 8 software (Axon Instruments). Recordings were performed with solutions containing equal concentrations of K+ applied to the bath and recording pipettes. The solution contained (mM) 10 KCl, 105 potassium gluconate, 5 KF, 20 potassium pyrophosphate, 0.1 sodium vanadate, 5 EGTA, 5 glucose, and 10 HEPES (pH = 7.4). Pyrophosphate and vanadate prevent channel rundown, and their concentrations in the patch solution have been shown to have no effect on Kir6.2 channels (29, 37). With this solution, a 10–15% rundown of the WT channels was seen in 10 min, a period that was sufficient for most of our experimental protocol.

Single-channel conductance was measured with slope command potentials from 100 to –100 mV. The open state probability (Popen) was calculated by first measuring the time tj spent at current levels corresponding to j = 0, 1, 2,... N channels open, based on all evident openings during the entire period of record (37, 38, 41). Popen was then obtained as Popen = ({sum}Formulatj j)/TN, where N is the number of channels active in the patch and T is the duration of recordings. Popen values were calculated from one to three stretches of data of 20 s acquired with Clampex 8 (Axon Instruments).

Low-pH exposures were carried out with the same bath solutions that were titrated to various pH levels as required by the experimental protocols. For ATP exposures, pH levels were adjusted after appropriate ATP concentrations were made in each solution. To avoid ATP degradation, all ATP-containing solutions were made immediately before experiments and used for no longer than 3 h. Because the variation of Cl– concentration in solutions was rather small, the resulting liquid junction potential was <1 mV according to the Henderson equation (see Ref. 2) and was therefore not corrected.

Currents were measured at peak inward rectification with Clampfit 8 software (Axon Instruments). A value was obtained after an average of eight consecutive records. Leak currents were removed from the data by subtracting the residue currents when the Kir6.2 channel was completely inhibited. The pH-current relationship was described with the Hill equation y = 1/[1+(pH/pKa)Formula], where pKa is the pH level for midpoint channel activation and nH is the Hill coefficient. Similarly, the ATP-current relationship was produced with the ATP concentration for half-channel inhibition (IC50) calculated. All data used for fitting were averaged first, and the mean values were then fitted with Hill equations. We did not attempt to fit data from each individual patch, because the difference between these two fitting methods was minimal (37, 38, 41). Data are presented as means ± SE. Differences in means were tested by ANOVA or Student's t-test and were accepted as significant if P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of R371H and P266T variants on baseline channel activity.
Single-channel activity was studied in inside-out patches with symmetrical concentrations of K+ (145 mM, pH 7.4) applied to both sides of patch membranes. Command potentials from –100 mV to 100 mV were applied to these patches from a holding potential of 0 mV. Under this condition, inward rectifying K+ currents were seen in both R371H and P266T mutations expressed with the SUR2A subunit. The slope conductance was 75.8 ± 1.5 pS (n = 13 channels from 6 patches) for the R371H variant and 76.4 ± 2.4 pS (n = 8 channels from 6 patches) for the P266T variant (Fig. 1A). No statistical difference was found between WT and these two variants (P > 0.05), indicating that these mutants still express functional currents although they occur at two highly conserved regions in the Kir6.2 subunit.


Figure 1
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  		Fig. 1. Baseline single-channel properties. Single-channel recordings were performed in inside-out patches with symmetric concentrations of K+ (145 mM) applied to both sides of the patch membrane. A: unitary conductance was measured with a ramp command potential (membrane potential, Vm) from –100 to 100 mV at pH 7.4. Straight lines indicate a slope conductance of 80 pS in the R371H variant (left) and 75 pS in the P266T variant (right). B: channel open state probability (Popen) was measured at Vm of –80 mV. The R371H variant (top) showed baseline Popen of 0.418. In comparison, the baseline Popen of P266T (middle) and heteromeric wild-type (WT)/P266T (bottom) channels were much higher, although the number of channels was lower. At left: C, closure; 1, 1st open state; 2, 2nd opening; 3, 3rd opening. C: statistically, the baseline Popen of the P266T and WT/P266T channels was significantly higher than those of the WT and R371H variant channels (***P < 0.001), whereas no significant difference was found between the WT and R371H variant channels. Data are means ± SE (n = 8–13).

 
Popen was measured at a membrane potential of –80 mV in the absence of ATP (pH 7.4). The baseline Popen of the P266T mutation was almost twice as high as that of the WT channel (P < 0.001), whereas no significant difference in Popen was found between the R371H and WT channels (P > 0.05; Fig. 1, B and C). The enhanced baseline channel activity was not only limited to the homomeric P266T. Coexpression of P266T with WT Kir6.2 also led to a marked increase in Popen (Fig. 1C). Because the Popen of R371H did not change, heteromeric expression was not attempted.

In these single-channel studies, we realized that the chances of seeing functional currents of the homomeric P266T mutant were much lower than those of seeing the WT channels, suggesting that the P266T variation may result in a reduction in channel density in plasma membranes. To test this possibility, we studied whole cell currents in single-electrode voltage clamp with symmetrical K+ concentrations (145 mM, pH 7.4). Current density was calculated by dividing the currents by the whole cell capacitance. We found that the current density of P266T was only half that of the WT channels (P < 0.001), although the current density of homomeric R371H and heteromeric WT/P266T did not show significant difference from the WT channels (P > 0.05; Fig. 2B). Channel density was also calculated by normalizing the current density with baseline Popen. Both homomeric P266T and heteromeric WT/P266T showed marked reductions in channel density (P < 0.001 and P < 0.01, respectively, compared with WT channels). The homomeric P266T channel density was only ~20% of that of WT channels (Fig. 2C).


Figure 2
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  		Fig. 2. Channel density. A: whole cell currents were recorded with a bath solution containing 145 mM K+. Pulse command potentials (–100 and 60 mV) were applied to the cell from a holding potential of 0 mV. Under this condition, currents with inward rectification were seen in both WT and mutant channels. Whereas the amplitude of the inward rectifying currents of the R371H and WT/P266T was similar to the WT channel, the P266T currents were much smaller than the WT currents. B: current density was measured by dividing the inward rectifying currents by whole cell capacitance. The current density of the homomeric P266T channel was lower than that of the WT channels (**P < 0.01), whereas the current density of the homomeric R371H and heteromeric WT/P266T did not show significant difference from the WT channels (P > 0.05). C: channel density was obtained by normalizing the current density with baseline Popen (note that single-channel conductance did not show any difference among these channels). Channel density of P266T and WT/P266T was only ~20% (***P < 0.001) and ~30% (**P < 0.01) of that of the WT channel, respectively. Data are means ± SE (n = 6–15).

 
Loss of allosteric modulation of ATP sensitivity by pHi in R371H and P266T variants.
ATP sensitivity was studied in WT channels and both mutant channels in giant inside-out patches with an equal concentration of K+ (145 mM) applied to both sides of plasma membranes. Command potentials from –100 mV to 100 mV were applied to these patches from a holding potential of 0 mV. Exposures of the internal patch membranes to different ATP concentrations produced a concentration-dependent inhibition of the inward rectifier currents (Fig. 3, A–D). The ATP-current relationship was described with the Hill equation (see MATERIALS AND METHODS). At pH 7.4, the ATP concentration for 50% current inhibition (IC50) was 25 µM [nH = 1.0, n = 16 (number of patches)] for the homomeric R371H channel, which was very close to that for the WT channel (IC50 = 22 µM, nH = 1.2, n = 11). ATP sensitivity was also studied in the heteromeric WT/R371H channel. A clear reduction in ATP sensitivity was seen (IC50 = 100 µM, nH = 1.1, n = 11; Fig. 3E).


Figure 3
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  		Fig. 3. Dose-dependent inhibition of R371H and WT/R371H currents by ATP. Currents were recorded in giant inside-out patches with symmetric concentrations of K+ (145 mM). Ramp command potentials from 100 to –100 mV were applied to the patches from a holding potential of 0 mV. Dose responses to different ATP concentrations ([ATP]) were studied at intracellular pH (pHi) 7.4 and 6.8. The midpoint inhibition of the R371H currents occurred at ~30 µM ATP at both pHi 7.4 (A) and pHi 6.8 (B). Note that 8 superimposed traces are shown in each panel. The WT/R371H current also showed similar ATP sensitivity at pHi 7.4 (C) and pHi 6.8 (D), with 50% current inhibition at ~100 µM. E: current amplitude measured at –100 mV was expressed as a function of [ATP] with the Hill equation. IC50 was 22 µM at pHi 7.4 and 70 µM at pHi 6.8 for the WT channel (dashed lines). However, the ATP sensitivity of homomeric R371H remained the same at these pH levels. A similar phenomenon was also seen in the heteromeric WT/R371H channel, although the curves shifted to higher ATP levels. See text or Supplemental Table S1 for IC50 and Hill coefficient (nH) values.

 
Because the ATP sensitivity of WT KATP channels is allosterically modulated by pHi (37), we examined such an allosteric modulation in the R371H variants. When the internal solution became acidic (pHi 6.8, seen in severe intracellular acidosis), ATP sensitivity was markedly reduced in the WT Kir6.2/SUR2A channel (IC50 = 70 µM, nH = 1.2, n = 6; Fig. 3E), consistent with our previous observations (37) on the Kir6.2/SUR1 and Kir6.2{Delta}C36 channels. Strikingly, the allosteric effect was completely eliminated in the homomeric R371H channel. The ATP sensitivity of the channel at pHi 6.8 (IC50 =2 5 µM, nH = 1.2, n = 11) was identical to that at pHi 7.4 (Fig. 3E). A similar observation was obtained from the heteromeric WT/R371H channel (IC50 = 120 µM, nH = 1.1, n = 11; Fig. 3E).

We failed to complete tests of ATP sensitivity in patches obtained from cells transfected with homomeric P266T. This was due to not only its low expression density but also the rapid channel rundown. Similar to WT/R371H, the heteromeric WT/P266T channel had low ATP sensitivity at pHi 7.4, with IC50 of 120 µM (nH = 1.2, n = 6; Fig. 4, A and C). The ATP sensitivity of the heteromeric WT/P266T channel changed very little at pHi 6.8 (IC50 = 140 µM, nH = 1.4, n = 8; Fig. 4, B and C). Thus the P266T variant also lost the allosteric modulation of its ATP sensitivity by intracellular protons.


Figure 4
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  		Fig. 4. ATP sensitivity of the WT/P266T channel. A and B: ATP-current relationship was studied under the same conditions as Fig. 3. The midpoint current inhibition occurred at ~100 µM ATP. C: ATP-current relationship was plotted with the Hill equation. Although the WT/P266T channel showed lower ATP sensitivity than the WT channel, ATP sensitivity was unaffected by changing pHi. See text or Supplemental Table S1 for IC50 and nH values.

 
Control experiments were done in another common Kir6.2 polymorphism, I337V. A clear difference in ATP sensitivity was seen at these pH levels (IC50 = 30 µM at pHi 7.4, IC50 = 80 µM at pHi 6.8; see Supplemental Table S1, available at the Physiological Genomics web site).1

Disruption of ATP-dependent modulation of pH sensitivity in R371H and P266T mutants.
In addition to ATP, WT KATP channels are sensitive to pHi. They are directly activated by intra- but not extracellular protons, and pHi sensitivity is subject to allosteric modulation by intracellular ATP (37, 38). To elucidate how such a modulation is affected by the R371H and P266T polymorphisms, we studied pHi sensitivity in inside-out patches. Exposures of internal patch membranes to a perfusate with various pH levels augmented the currents in the moderate acidic pHi range, and the currents were inhibited at extremely acidic pHi levels. In the homomeric R371H channel, peak activation occurred at pHi 6.2 in the presence of either 0.3 mM or 1.0 mM ATP (Fig. 5, A and B). pHi sensitivity of the heteromeric WT/R371H channels was not affected by these concentrations of ATP, although the peak activation shifted to pHi 5.9 (Fig. 5, C and D). The pHi-current relationship was described with the Hill equation. Because the channel inhibition at extremely acidic pHi is not seen in whole cell recordings and appears to be caused by channel rundown (37–39), it does not seem to play a role in cellular function. Therefore, we only focused our data analysis on pH-dependent channel activation. Consistent with our previous reports (37), ATP modulated the pH sensitivity of WT Kir6.2/SUR2A channels. In the presence of 0.3 mM ATP, the pHi level for 50% current activation (pKa) was 6.60 in the WT channels with nH = 3.2 (n = 7; Fig. 5E). The pHi-current relationship shifted toward lower pH levels in the WT channels when there was 1.0 mM ATP in the internal solution (pKa = 6.36, nH = 3.2, n = 10). Such an allosteric modulation, however, was not seen in the R371H mutant. Its pKa values were 6.64 (nH = 3.0, n = 17) with 0.3 mM ATP and 6.67 (nH = 3.0, n = 14) with 1.0 mM ATP (Fig. 5E). A similar phenomenon was seen in the heteromeric WT/R371H channel (pKa = 6.7, nH = 2.5, n = 15 with 0.3 mM ATP; pKa = 6.73, nH = 2.5, n = 11 with 1.0 mM ATP; Fig. 5E), suggesting that the allosteric effect of ATP on pHi sensitivity is disrupted in R371H mutant channels.


Figure 5
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  		Fig. 5. Effects of R371H variants on pH sensitivity. A: current response to acidic pHi was studied by exposing the internal surface of the patch membrane to a perfusate with various pH levels as indicated at top. In the presence of 0.3 mM ATP, evident increase in the current amplitude was seen at pHi 7.1. The maximal activation was reached at pHi 6.2. Further decrease in pHi caused a rapid inhibition of the currents. B: a similar pH response was observed in the presence of 1.0 mM ATP. A high ATP concentration failed to change the pH sensitivity, and the peak current activation also occurred at pHi 6.2. C and D: similarly, the heteromeric WT/R371H currents did not show ATP-dependent modulation of pH sensitivity, with maximum current activation at pHi 5.9 and 6.2 with 0.3 and 1.0 mM ATP, respectively. Note that 8 superimposed traces are shown in each panel. E: pH-current relationship was described with the Hill equation. With 0.3 mM ATP, the midpoint pH for channel activation (pKa) was pHi 6.60 for WT, pHi 6.64 for R371H, and pHi 6.70 for WT/R371H. The pKa values increased to pHi 6.36 in WT but showed almost no changes in R371H and WT/R371H channels (6.67 and 6.73, respectively) in the presence of 1.0 mM ATP.

 
Like the WT and R371H mutant channels, the heteromeric WT/P266T channels displayed a biphasic response to acidic pH (Fig. 6). With 0.3 mM ATP, the WT/P266T variant showed pKa of 6.51 and nH of 2.1 (n = 6; Fig. 6B). When ATP concentration increased to 1.0 mM, the pKa value was moderately reduced to 6.42 with nH = 2.9 (n = 7), a change of 0.09 pH units compared with 0.24 pH units in the WT channel (see Supplemental Table S2). Thus these results indicate that the P266T polymorphism also impairs the allosteric modulation of pH sensitivity.


Figure 6
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  		Fig. 6. Response of the WT/P266T channel to pHi. pH sensitivity of the WT/P266T channel was studied under the same experimental conditions as in Fig. 5. A and B: a similar biphasic response was seen, with the maximal activation occurring at pHi 5.9 with 0.3 and 1.0 mM ATP. C: the Hill equation was used to describe the pH-current relationship. The pKa of the WT/P266T channel was pHi 6.51 (nH = 2.1) and 6.42 (nH = 2.9) with 0.3 and 1.0 mM ATP, respectively,

 
In contrast to R371H and P266T, the I337V mutant showed pKa of 6.64 in the presence of 0.3 mM ATP and pKa of 6.40 with 1.0 mM ATP, a 0.24-pH unit change identical to the WT channel (Supplemental Table S2).

Effects on outward currents.
The WT KATP channels have weak inward rectification, allowing substantial outward currents. Therefore, we measured the ratio of outward currents to inward currents of the R371H and P266T mutants. Figure 7 shows the comparison of the current ratios. Although the current ratios were almost the same at pHi 7.4 (Fig. 7A), differences were found at pHi 6.8. The current ratio of the P266T mutant was significantly lower than that of the WT channel (Fig. 7B), indicating that outward currents of the P266T mutant were more sensitive to acidic pHi than inward currents. A similar decrease in outward currents of the P266T mutant was seen in the presence of 10 µM ATP. Indeed, the effect on outward currents was independent of ATP, as similar levels of reduction in outward currents were observed with ATP at various concentrations (P > 0.05), although the current ratio was slightly lower with 300 µM ATP (Fig. 7C). In contrast to P266T, the ratios of outward vs. inward currents remained the same in R371H and WT/R371H channels (Fig. 7, A and B).


Figure 7
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  		Fig. 7. Effect of P266T and R371H mutations on outward currents. A: ratio of outward currents (I) to inward currents was studied in WT and mutant channels. No significant difference was found with (10 µM) and without ATP at pHi 7.4. B: at pHi 6.8, the current ratio was significantly lower in the WT/P266T than in the WT channel (*P < 0.001). No significant difference was seen in other channels. C: reduction in current ratio at pHi 6.8 was independent of ATP, as it occurred to almost the same degree at various ATP levels.

 
We also examined ATP and pH sensitivities of the outward currents. Compared with inward currents, the outward currents of P266T showed similar ATP and pH sensitivities, although there were small changes in nH values (Fig. 8). The ATP and pH dose-response curves of the WT, R371H, and WT/R371H were nicely described with the same ATP IC50, pKa, and nH values that were used for inward currents (see Supplemental Tables S3 and S4). These results thus suggest that the P266T and R371H mutations cause abnormalities in allosteric regulation of channel activity by ATP and pH in both inward and outward currents.


Figure 8
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  		Fig. 8. ATP and pH sensitivities of WT/P266T outward currents. A: outward currents were measured and the ATP dose-response relationship studied as in Fig. 4. These ATP-current relationships were identical to those of inward currents except in WT/P266T at pHi 6.8, with a decrease in IC50 (by 20 µM) and an increase in nH value (by 0.2) over those for inward currents. B: pH-current relationship was studied as in Fig. 6. pKa of the WT/P266T channel was pH 6.55 (nH = 2.0). All others were the same as for the inward currents.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Cardiovascular diseases are the leading cause of death. Among all deaths from cardiovascular diseases, ~50% belong to SCD, seen mostly in ischemic myocardial infarction and heart failure (33). The key contributor to SCD is lethal ventricular tachyarrhythmias, particularly ventricular fibrillation (36). It is widely accepted that instability of membrane electrophysiological properties of myocardium leads to ventricular arrhythmias and thus SCD. The KATP channels regulate membrane potentials and myocardial excitability (11, 28). Activity of these channels is controlled by several metabolites, allowing them to play a role in cellular responses to metabolic stress. Indeed, previous studies have shown that repetitive exposures to sublethal ischemia protect the heart from a lethal ischemic insult, known as cardiac ischemic preconditioning, in which the mitochondrial KATP channels are known to play an important role and the sarcolemmal channels are also involved (15, 32). Under hypoxic ischemic conditions, KATP channels are activated. The activation of the KATP channels can shorten the time for Ca2+ influx via L-type Ca2+ channels and increase the time of Ca2+ extrusion through the Na+/Ca2+ exchange system (5). The decrease in Ca2+ influx tends to reduce mechanical contraction and energy consumption when oxidative phosphorylations are inhibited during hypoxic ischemia. Strong evidence comes from Kir6.2-knockout mice, in which several survival advantages are lost, such as exercise training of myocardium, prevention of arrhythmia caused by catecholamine challenge, and hypoxic myocardial protections (16, 19). The protective effect of KATP channels on myocardium is further supported by administration of channel blockers and openers. A sulfonylurea KATP channel antagonist, glibenclamide, attenuates the shortening of action potential duration and impairs functional recovery of the ventricle after reperfusion (5). Consistently, KATP channel openers such as bimakalim accelerate the rate and extent of the action potential shortening and improve the functional recovery (40). Experimental evidence has suggested that activation of sarcolemmal KATP channels may not always lead to a beneficial effect during cardiac ischemia, as pinacidil is found to have a proarrhythmia effect (4, 6). Because the primary target of pinacidil is the vascular isoform (Kir6.1/SUR2B), this effect may be secondary to vasodilation and systemic blood pressure changes or a result of enhanced catecholamine release (23).

The sarcolemmal KATP channels are inhibited by intracellular ATP and augmented by intracellular protons (7, 21, 38). Thus channel activation can be produced by the decrease in pHi and/or ATP concentration during hypoxic ischemia. In addition to these direct effects, ATP and protons interact with each other through the allosteric mechanism that allows an even better control of channel activity according to metabolic states: a decrease in pHi can not only activate the channel directly but also relieve the channel inhibition by ATP. pH-dependent channel activation is further enhanced with a drop in ATP concentration, as the pH-current relationship shifts to higher pH levels with lower ATP levels (37). With such an allosteric regulation, KATP channel activity tends to change moderately with enhanced metabolic activity during exercise or sublethal hypoxia. The channels can be strongly activated with severe metabolic challenges such as lethal myocardial ischemia. Such hierarchical activations of the WT KATP channels are mostly lost in the polymorphic R371H and P266T channels, although they remain to respond to intracellular ATP and pH alone. Apparently, the loss of allosteric modulation in the polymorphic R371H and P266T channels eliminates the beneficial effect on channel activation during severe metabolic stress.

Of particular importance is that the disruption of the allosteric effect was observed in both heteromeric R371H/WT and P266T/WT channels, as they are the genotype found in SCD patients (14). Heteromeric R371H/WT channels showed ATP sensitivity that was clearly different from that of either homomeric R371H or homomeric WT channels. We modeled the potential effect produced by their expression as two separate functional entities by a mathematical sum in various ratios and found no evidence for such functional expression. Therefore, it is very likely that impairment of the channel modulation by ATP and pH occurs in heteromeric R371H/WT and P266T/WT channels. How the allosteric modulation is affected by the R371H and P266T variations is unclear. Because this area of the COOH terminus is known to be involved in ATP sensitivity (8), it is possible that a replacement of amino acid residues in the two conserved regions may prevent the conformational change necessary for allosteric modulation.

Another finding from the present study is the great reduction in channel density of the P266T variant. The channel density of the homomeric P266T is only ~20% of the WT channel. This level, which was measured in cells showing positive expression, seems to be overestimated, as functional currents were not detected in most cells. The channel density remains low even with heteromeric expression of the P266T/WT channel. Although the enhanced basal channel activity may compensate functional channel activity to a small degree, the poor functional expression is likely to contribute to the adverse consequences in patients who die of SCD (14).

P266T also shows a significant reduction in outward currents at acidic pHi. The ratio of outward currents to inward currents is ~20% lower than that of the WT channel. This effect does not seem to be mediated by changes in the ATP and pH dose-response relationship. We speculate that the mechanism for inward rectification might be affected by the P266T mutation. Further experiments are needed to understand the molecular basis for such an effect. Although the current ratios did not show significant changes in R371H and WT/R371H, their ATP and pH sensitivities are identical to those of the inward currents. Similar phenomena were found in WT/P266T. Therefore, our results indicate that both inward and outward currents were affected by the P266T and R371H mutations.

Considering the unpredicted nature of SCD, the discovery of genetic links to the diseases is a promising approach to the early diagnosis of myocardial infarction, instantaneous administration of therapeutic modalities, and effective prevention of ventricular arrhythmias (10, 17). Jeron et al. (14) systematically studied all common single-nucleotide polymorphisms and point mutations in the Kcnj11 gene and found that other polymorphisms are not related to SCD, including the E23K that is known to be attributable to Type 2 diabetes (27). Although a number of genetic variations associated with SCD have been found over past years, including the P266T and R371H variants (1, 14, 18, 20, 24, 31), whether these genetic variants really underlie SCD depends on the demonstration of their functional consequences. In this regard, our present studies appear to constitute a significant step in the understanding of SCD. The determination of genetic links to myocardial infarction and SCD should provide important information not only to physicians but also to carriers, because this may motivate them to reduce potential risk factors for cardiovascular diseases by changing their living habits and lifestyles.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-058410 and HL-067890.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Susumu Seino at Kobe University in Japan for the gift of Kir6.2 cDNA and Dr. Joseph Bryan at Baylor College of Medicine for the gift of SUR2A cDNA.

Present address of X. Wang: Dept. of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: C. Jiang, Dept. of Biology, Georgia State Univ., 24 Peachtree Center Ave., Atlanta, GA 30302-4010 (e-mail:cjiang{at}gsu.edu).

* N. Cui, L. Li, and X. Wang contributed equally to this work (names listed in alphabetical order). Back

1 The Supplemental Material for this article (Supplemental Tables S1–S4) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00106.2005/DC1. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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