1School of Biomedical Sciences and 2Institute of Cardiovascular Research, University of Leeds, Leeds LS1 9JT, United Kingdom
Submitted 4 August 2003 ; accepted in final form 17 September 2003
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ABSTRACT |
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potassium channel; tandem P domain
Although arachidonic acid activation of TREK is completely occluded in hypoxia, no information is available regarding the potential interaction of intracellular pH and O2 availability. This is particularly important because intracellular acidosis is a key pathological feature of central ischemia. We show here that activation of hTREK1 channels by intracellular acidification is completely occluded at low PO2, consistent with the assumption that the mechanisms responsible for channel modulation by acidification, fatty acids, and O2 may demonstrate significant overlapping structural requirements. Unlike its murine counterpart, hTREK1 is inhibited by intracellular alkalinization. Furthermore, neither arachidonic acid nor hypoxia are able to occlude this reduction in channel activity, which is evoked by intracellular alkalinization. This finding leads us to conclude that there must be additional determinants required for channel closure during intracellular alkalosis. As a consequence of these observations, it appears certain that upregulation of hTREK1 activity (by either fatty acid or acidotic insult) during ischemia will not occur in the brain. Indeed, our data suggest that an inhibition of hTREK1 activity during situations of metabolic or respiratory alkalosis is likely to be more physiologically relevant in tissues where ambient PO2 is low.
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MATERIALS AND METHODS |
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Electrophysiology. Cells were grown for 24 h on glass coverslips before being transferred to a continuously perfused (5 ml/min) recording chamber (volume 200 µl) mounted on the stage of a Nikon TMS inverted microscope. For whole cell patch-clamp recordings, the standard perfusate was composed of (in mM) 135 NaCl, 5 KCl, 1.2 MgCl2, 5 HEPES, 2.5 CaCl2, 10 D-glucose, and 30 sucrose (pH 7.4). Whole cell K+ currents were recorded at room temperature (21 ± 1°C) with the use of a pipette solution composed of (in mM) 10 NaCl, 117 KCl, 2 MgCl2, 11 HEPES, 11 EGTA, 1 CaCl2, and 2 Na2ATP (pH 7.2). For experiments that employed static changes of intracellular pH, HEPES was substituted by either PIPES (pH 6.5) or Tris (pH 7.9). When filled with these solutions, pipettes were of resistance 57 M
. All solutions were bubbled with either N2(g) (relative hypoxic solutions) or medical air (control solutions) for at least 30 min before perfusion of cells. These maneuvers produced no shift in either bath pH or temperature. PO2 was measured (at the cell) by using a polarized carbon fiber electrode (19), and with use of the protocols described herein, relative hypoxia is defined as a PO2 of between 20 and 30 Torr. Bath temperature was monitored throughout by using a thermistor-based digital thermometer. Arachidonic acid (10 µM), ammonium chloride (20 mM), trimethylammonium (20 mM), and sodium propionate (10 mM) were applied via the perfusate (isosmotic substitution of NaCl as appropriate). Current recordings were made by using an Axopatch 200A amplifier and Digidata 1200 analog-to-digital interface (Axon Instruments, Foster City, CA).
To evoke whole cell K+ currents, we used standard ramp-step protocols (18). Briefly, cells were voltage-clamped at 70 mV and were ramped from 100 to +60 mV over 1 s. Cells were then sequentially stepped from the holding potential to 0 and +60 mV for 100 ms each. This protocol was repeated at a frequency of 0.1 Hz. Cell-attached recordings were obtained by using patch pipettes filled with standard extracellular solution. In these experiments, pipette potential was held at 0 mV, and 1-s voltage ramps (from +30 to 130 mV, which mimics the whole cell voltage protocol in which resting membrane potential was clamped to 70 mV) were applied at a frequency of 0.1 Hz. Data were acquired as for whole cell recordings. Data are presented as example traces together with means ± SE. Time series plots were constructed from the current amplitudes measured over the last 10 ms of the +60-mV step (whole cell) or at 5 ms before 130 mV was attained (cell attached). Outward currents in the whole cell configuration are represented by upward deflections, whereas, by convention, the reverse is true of cell-attached currents. Statistical comparisons were made by using paired or unpaired Student's t-test or ANOVA, as appropriate.
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RESULTS |
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Dynamic modulation of intracellular pH: whole cell recording of hTREK1 currents. Although static modulation of intracellular pH via controlling pipette pH provided novel baseline information concerning regulation of hTREK1 currents in the whole cell configuration (particularly that currents were suppressed by intracellular alkalinization), it was less useful as a means by which to study the polymodal nature of channel regulation. Thus a well-established method of producing dynamic alterations in intracellular pH, extracellular perfusion with weak acids (1) and bases (3), was adopted to study the influence of intracellular pH on hTREK1 channel activation by arachidonic acid (15) and inhibition by hypoxia (18). Figure 2A demonstrates that perfusion with 10 mM propionate evoked a rapid hTREK1 current activation, consistent with previous data obtained by using the murine TREK1 channel homologue (13). Conversely, perfusion with 20 mM ammonium chloride, a known intracellular alkalinizing agent (see, for example, Ref. 9) evoked rapid current inhibition (Fig. 2A); this observation is different from that previously reported from mTREK1 studies (13). Because the ammonium pulse protocol was invariably characterized by a slow transient response that resulted in a mild, but not full, reversal of the inhibitory effect (consistent with the cellular adaptation to alkalosis, which is seen with this agent in experiments that measure intracellular pH directly; see Ref. 9), application of the weak base trimethylamine (TMA) was also conducted (Fig. 2A). With the use of TMA, currents were again inhibited, confirming that hTREK1 channel inhibition was due to intracellular alkalinization. In paired experiments, propionate evoked a significant (P < 0.05) current augmentation to 130 ± 4% of control (n = 23), whereas ammonium and TMA significantly (P < 0.05) reduced the currents to 57 ± 3% (n = 6) and 52 ± 3% (n = 26) of control, respectively (Fig. 2B). These results indicate for the first time that intracellular alkalinization evokes hTREK1 current suppression.
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Modulation of the pH responses by relative hypoxia. We have recently shown that relative hypoxia is able to abolish completely the potential activation of hTREK1 by arachidonic acid, suggesting that ambient PO2 plays a pivotal role in dynamic modulation of these channels under physiological circumstances (18). Figure 3, A (current traces), B (time course of action), and C (mean effects), extends that previous observation to show that activation by intracellular acidification is completely abolished by simultaneous reduction in bath PO2 to 25 Torr. In this series of experiments, propionate evoked a rise in mean current density from 228 ± 31 to 293 ± 33 pA/pF (P < 0.005, n = 9, Student's paired t-test), whereas reducing bath PO2 in the continued presence of propionate resulted in a dramatic reversal of this activation and suppressed currents to 168 ± 18 pA/pF; this effect was freely reversible upon wash. The normalized current density data are shown in Fig. 3C, which also serves to highlight that, in addition to the complete blockade of acid activation, relative hypoxia was able to evoke further current suppression to a level significantly below that seen before propionate addition (P < 0.02).
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To ascertain the nature of the hypoxic occlusion of acid activation and the lack of hypoxic modulation of alkali inhibition, we conducted two series of experiments in which the order of addition of modulators was reversed (Figs. 5 and 6). In both series, reduction of bath PO2 to 25 Torr resulted in significant and sustained current density suppression from 532 ± 105 to 427 ± 87 pA/pF (propionate experiments, n = 11, P < 0005; Fig. 5, A, B, and C) and from 435 ± 64 to 326 ± 44 pA/pF (TMA experiments, n = 9, P < 0.002; Fig. 6, A, B, and C); proportionally, these inhibitory effects were not different from the hypoxic responses exemplified in Figs. 3 and 4 and earlier (18). Importantly, coapplication of propionate with relative hypoxia was without further effect (current density = 426 ± 90 pA/pF; Fig. 5). However, and by marked contrast, coapplication of 20 mM TMA with relative hypoxia further significantly reduced current density to 122 ± 23 pA/pF (P < 0.0002). The mean, normalized data are plotted in Figs. 5C and 6C and indicate that the order of modulator addition is not important to the ability of relative hypoxia to occlude differentially the activation by intracellular acidification.
The transient activation noted in Fig. 5B coincided with a bath rewarming transient, which can occur when two gassed solutions are perfused sequentially (bath temperature was monitored constantly by using a bath thermistor). Such transients were of variable magnitude and were also seen during equivalent gas-to-gas transitions exemplified in Figs. 6B (small transient), 9B, and 10B.
Interaction of the influences of intracellular pH, hypoxia, and arachidonic acid. Arachidonic acid is a potent activator of both hTREK1 (18) and mTREK1 (23). However, similar to the effect documented herein for intracellular acidification, arachidonic acid activation is completely blocked when PO2 is reduced to below 60 Torr (18). To understand more completely the level of interaction of these important channel modulators, we employed a combination of all three maneuvers. Addition of 10 mM propionate elicited the expected current density augmentation from 359 ± 57 to 425 ± 76 pA/pF (n = 6; Fig. 7). A maximal concentration of arachidonic acid (10 µM) (18) evoked a further increase to 557 ± 122 pA/pF (Fig. 7). Importantly, coapplication of relative hypoxia with both of these activators completely abolished their combined activating influences and suppressed the current density to 195 ± 75 pA/pF (Fig. 7).
Addition of 20 mM TMA evoked the expected inhibition of current density from 423 ± 38 to 236 ± 35 pA/pF (Fig. 8). As in the propionate experiments, arachidonic acid was able to evoke a robust increase in current density to 567 ± 70 pA/pF in the presence of TMA (Fig. 8). Coapplication of relative hypoxia with both agents completely abolished the arachidonic acid activation and reduced the current density to 247 ± 44 pA/pF (Fig. 8; n = 8), a level not significantly different from that seen in the presence of TMA alone. The mean, normalized current densities are plotted in Figs. 7C (propionate) and 8C (TMA).
After dynamic change of intracellular pH with propionate and TMA, changing the order of addition of arachidonic acid and hypoxia produced broadly the same data as those described above, with two minor differences. Thus, although hypoxia completely abolished the propionate activation (Fig. 9), arachidonic acid coapplication consistently resulted in a small transient increase in current density followed by a sustained suppression (see Fig. 9). Relative hypoxia reduced the propionate response from 440 ± 78 to 245 ± 54 pA/pF, which was then further reduced by arachidonic acid to 165 ± 49 pA/pF (P < 0.01, n = 8).
Consistent with earlier data, relative hypoxia had an additive effect on TMA-induced intracellular alkalinization (Fig. 10). In contrast to the inhibitory effect of arachidonic acid that was observed after hypoxic acidification, during hypoxic alkalinization, arachidonic acid evoked no further sustained effect (Fig. 10) with current density changing from 156 ± 32 to 154 ± 35 pA/pF (n = 11). As mentioned earlier, the transients noted in Figs. 9B and 10B represent bath rewarming transients that can occur upon gas-to-gas transitions. The mean current data for the intracellular acidification and alkalinization are plotted in Figs. 9C and 10C, respectively.
Dynamic modulation of intracellular pH: cell-attached recording of hTREK1 currents. As a method by which to study further the modulating influences of intracellular pH, arachidonic acid, and relative hypoxia but in a more physiologically appropriate setting, experiments were conducted using the cell-attached configuration. In this configuration, hTREK1 currents were increased by intracellular acidification from 22.8 ± 3.9 to 32.8 ± 4.9 pA (Fig. 11; n = 6, P < 0.05). Channel activity was further enhanced by coapplication of arachidonic acid to 47.3 ± 8.1 pA (Fig. 11). As for whole cell experiments, these additive effects were occluded by relative hypoxia such that mean currents were reduced to 29.1 ± 4.6 pA, a value not significantly different from control (P > 0.1). Figure 12 shows for the first time that although unstimulated cell-attached currents were small, relative hypoxia was able to reduce them significantly from 22.4 ± 2.6 to 19.4 ± 2.2 pA (n = 8; P < 0.01). However, and consistent with the whole cell data, in the presence of relative hypoxia, neither propionate nor subsequent addition of arachidonic acid could activate the current from this suppressed level (Fig. 12).
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Figure 13A, top, shows again that although resting cell-attached hTREK1 currents were small, intracellular alkalinization significantly reduced them from 21.3 ± 3.2 to 17.2 ± 3.2 pA (P < 0.05, n = 7). Furthermore, coapplication of 10 µM arachidonic acid evoked a robust increase to 86.7 ± 31.5 pA that was completely abolished during relative hypoxia. This hypoxic challenge reduced the current to 28.3 ± 5.3 pA, a value not significantly different from control (Fig. 13C; n = 7, P > 0.1). Reversal of the order of modulator addition in the cell-attached mode produced results predicted by, and compatible with, those observed with whole cell recording (Fig. 14). Thus, in the presence of relative hypoxia, cell-attached currents were not significantly affected by sequential addition of TMA and arachidonic acid (Fig. 14).
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DISCUSSION |
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Modulation of intracellular pH. Activity of hTREK1 is modulated by intracellular pH at values that could be considered to bracket the full pathophysiological range (pH 6.5 to 7.9). Thus, in addition to the previously documented activation of mTREK1 by intracellular acidification (corroborated here in hTREK1, see Figs. 1 and 2), our data show that intracellular alkalinization is a potent channel inhibitor (Fig. 2). Such suppression of channel activity has not been reported for mTREK1, and where it has been studied, inhibition was complete as pH was raised above 7.4 (13). Strong supporting evidence for depression of hTREK activity by intracellular alkalinization comes from the data employing ammonium and TMA, which show that dynamic suppression of hTREK1 activity is apparent often only seconds after perfusion with weak base (Figs. 4, 6, 8, and 10). Presently, there is no clear structural explanation for the significant discrepancy between the hTREK data presented herein and the previously documented mTREK data (13). Indeed, the only plausible site of action would be at His72 (in hTREK1), which is a glutamate in mTREK. Although the pKa of histidine makes it a more attractive candidate than glutamate for sensing changes in pH in the alkaline range, this residue is predicted to be extracellular and would not, therefore, respond to the intracellular alkalinization evoked by TMA or ammonium.
We employed three independent maneuvers to produce intracellular alkalinization, and with all it is not possible to be sure of the absolute pH values attained. However, by deferring to data from measurement in other cell types, it might be reasonable to assume that extracellular perfusion with 20 mM of either ammonium or TMA would elicit a pH rise of no more than 0.2 units (1, 3). On the basis of this knowledge, it is clear that alkalinization to levels associated with mild pathology (such as, for example, the respiratory alkalosis associated with rapid ascent to altitude or metabolic alkalosis induced by chronic vomiting) will be sufficient to suppress channel activity and thus depolarize cells in which resting membrane potential is largely dependent on TREK1 activity. Channel activity can also be mildly, but significantly, suppressed by intracellular alkalinization in the cell-attached configuration (Fig. 13), lending further weight to the argument that inhibition by increased pH may be of physiological relevance.
Another important regulatory influence is exemplified in Fig. 1, in which channel activity is shown to increase rapidly after transition from the cell-attached to the whole cell configuration at all intracellular pH values employed in this study. One possible explanation for such a phenomenon is that channel activity is under tonic inhibitory influences that are dependent on interactions with the cytoskeleton. If this were the case, we would predict that channel activity would also be much larger in inside-out patches than in cell-attached patches, a notion fully supported by the previous data garnered from mTREK1 and consistent with the knowledge that channel activity is strongly influenced by both mechanical (mTREK1) and chemical (mTREK1 and hTREK1) membrane distortion. Indeed, it is plausible that inhibition by alkalinization may be most important when channels are "preactivated," as they are in the whole cell configuration and as they would be under conditions of stress, such as during membrane deformation.
Modulation of the pH responses by hypoxia. Channel activation by intracellular acidification has been previously documented for mTREK1 (13) in normoxia (air-equilibrated perfusion solutions), and those observations are now extended for hTREK1 in whole cell (Figs. 3, 7, and 9) and cell-attached (Fig. 11) modes. We have previously demonstrated that the potent TREK1 activator arachidonic acid is unable to augment currents when PO2 is reduced below 60 Torr (18). Figure 3 demonstrates clearly that such dependence on ambient PO2 holds true for activation by intracellular acidification. This is a very important observation because it provides clear and further evidence against the idea that TREK1 activation in neurons within the CNS could mediate a hyperpolarization (and, therefore, provide neuroprotection) during ischemic insult by being activated by acidosis and/or arachidonic acid at PO2 values typical in the human brain in either health or disease. The idea that intracellular acidosis is not an activating influence at such low PO2 values is reinforced by the data in Figs. 5 and 12, which show a complete lack of activation by acid during relative hypoxic perfusion.
Although acidification cannot provide activation when PO2 is low, the converse does not hold true of intracellular alkalinization. Thus relative hypoxia was unable to reverse the inhibitory effect of TMA. Indeed, hypoxia is an inhibitory factor that is additive to intracellular alkalinization (Fig. 4). Such a conclusion is fully supported by the data in Fig. 6, which demonstrate that TMA is still able to elicit robust channel inhibition even when the bath PO2 is as low as 25 Torr. The stark contrast between the interactions of either acidification or alkalinization with hypoxia has at least two important consequences. First, the requirements for acid sensing and base sensing in the physiological range may reside in separate structural domains of the hTREK1 channel protein. Second, in the brain where PO2 is persistently very low, increased pH might be a key regulatory influence on hTREK1 activity and, by inference, of excitability of neurons in which it is expressed. Potentially, the conserved run of basic residues (RRRL, at positions 329 to 332 in the COOH terminal) might serve as a base sensor, since the pKa values are in the physiological range. If this were the case, the differences observed between hTREK1 and mTREK1, in terms of sensitivity to intracellular alkalosis, must be due to differences in experimental and/or expression protocols employed by the different laboratories.
Potential physiological roles. Although inhibition by intracellular alkalinization is of potentially greater importance to TREK1 channel regulation than acidification in tissues where PO2 is low, it is presently impossible to predict how such a potent inhibitory influence might be of physiological importance. However, it is tempting to speculate that in CNS neurons, where TREK1 expression is a major influence on resting membrane potential, respiratory or metabolic alkalosis will cause hyperexcitability. Alkalosis has several effects on the CNS but, in general, evokes increased activity, often leading to aberrant afferent ("pins and needles") and efferent (muscle twitch) firing. Although regulation of hTREK1 activity is unlikely to account fully for such phenomena, it may account for a component of such physiological responses. Equally, respiratory alkalosis is a characteristic of rising to high altitude as carotid body hypoxic drive to the medullary respiratory centers promotes hyperventilation and, therefore, removal of CO2. Under such circumstances, hyperexcitability of descending inhibitory neurons to the respiratory center may temper the hyperventilation and prevent further alkalosis during a sustained visit to altitude. If this is true, TREK1 modulation may be involved in the acclimatization response, for example. In this regard, it is interesting to note that hTREK1 predominately colocalizes with GABAergic neuronal markers in inhibitory interneurons (5), suggesting that TREK1 may be involved in controlling excitability via removal of tonic inhibitory influences during alkalosis. Where studied, it appears that expression of hTREK1 mRNA in nonneuronal tissues is of significance only in the early gastrointestinal tract (15). Our data would suggest, therefore, that hTREK1 in the stomach and small intestine would be expected to behave rather differently than central neuronal hTREK1, since ambient PO2 in these tissues is much higher than in brain. Of course, we also cannot be certain that hTREK in HEK-293 cells will behave similarly in human neurons, where it is natively expressed. However, until such tissue becomes available, expressing human channels in recombinant systems is a reasonable compromise for the study of human central neuronal ion channel function.
In addition to studying the effects of relative hypoxia on pH sensing by hTREK1, this study also sought to investigate the interdependency of these factors with arachidonic acid. This was principally because such data would provide insight into the potential mechanisms of channel modulation and their functional coincidence. With the knowledge from previous work (for mTREK1 at least) that the structural requirements for activation by acidification and arachidonic acid overlap at the amino acid residue E306 (7) together with the information that acid and base sensing do not, our data support strongly the idea that the mechanism for hypoxic inhibition is via functional occlusion of the acid/fatty acid sensory component specifically rather than via a "global" decrease in open-state probability in general. If the latter were the case, hypoxia should be able to occlude all regulatory signals, which it clearly does not.
In conclusion, our data indicate that at PO2 values typically measured in the human brain, hTREK1 cannot be activated by arachidonic acid or intracellular acidosis. Conversely, inhibition by intracellular alkalosis is unaffected by low PO2. Taken together, these data suggest that the alkali-sensing element of the channel is distinct from the polymodal E306 residue (possibly in the run of high pKa arginine and lysine residues between positions 329 and 332 in the COOH terminus) and that hTREK1 channels may be more suited to regulating neuronal excitability during respiratory and metabolic alkalosis than during acidosis or ischemia.
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ACKNOWLEDGMENTS |
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This work was funded by The British Heart Foundation and The Wellcome Trust.
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FOOTNOTES |
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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.
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