Polymodal regulation of hTREK1 by pH, arachidonic acid, and hypoxia: physiological impact in acidosis and alkalosis

Paula Miller,1 Chris Peers,2 and Paul J. Kemp1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of the human tandem P domain K+ channel, hTREK1, is limited almost exclusively to the central nervous system, where ambient PO2 can be as low as 20 Torr. We have previously shown that this level of hypoxia evokes a maximal inhibitory influence on recombinant hTREK1 and occludes the activation by arachidonic acid; this has cast doubt on the idea that TREK1 activation during brain ischemia could facilitate neuroprotection via hyperpolarizing neurons in which it is expressed. Using both whole cell and cell-attached patch-clamp configurations, we now show that the action of another potent TREK activator and ischemia-related event, intracellular acidification, is similarly without effect during compromised O2 availability. This occlusion is observed in either recording condition, and even the concerted actions of both arachidonic acid and intracellular acidosis are unable to activate hTREK1 during hypoxia. Conversely, intracellular alkalinization is a potent channel inhibitor, and hypoxia does not reverse this inhibition. However, increases in intracellular pH are unable to occlude either arachidonic acid activation or hypoxic inhibition. These data highlight two important points. First, during hypoxia, modulation of hTREK1 cannot be accomplished by parameters known to be perturbed in brain ischemia (increased extracellular fatty acids and intracellular acidification). Second, the mechanism of regulation by intracellular alkalinization is distinct from the overlapping structural requirements known to exist for regulation by arachidonic acid, membrane distortion, and acidosis. Thus it seems likely that hTREK1 regulation in the brain will be physiologically more relevant during alkalosis than during ischemia or acidosis.

potassium channel; tandem P domain


THE TANDEM P DOMAIN potassium channel TREK1 is localized almost exclusively to the central nervous system (CNS), where it is believed to play a key role in setting the resting membrane potential of the neurons in which it is expressed (4, 12, 16, 17). Evidence also exists for its expression in the peripheral nervous system, in general and sensory neurons of the dorsal root ganglion in particular (12). On the basis of the ability of TREK to influence the resting membrane potential of neurons, coupled to evidence of its specific regulation by unsaturated fatty acids (4, 15, 23), membrane stretch (23), acidosis (12, 13), and inhalation anesthetics (22), it has been suggested that this background K+ channel may control neuronal excitability and play a neuroprotective role during brain ischemia, where local pH declines and arachidonic acid is released into the extracellular space (see, for example, Ref. 24). Further support for such an idea has come from the demonstration that the neuroprotective agent riluzole is a TREK1 activator (2, 15) and that arachidonic itself is neuroprotective (10). Compelling though this argument might have been, it was based exclusively on studies of TREK1 channel regulation at a partial pressure of O2 (PO2) of ~150 Torr (i.e., room air-equilibrated solutions). While this might be perfectly reasonable for ion channels that are expressed in the periphery [because PO2 is normally between 80 and 100 Torr and O2-sensitive K+ channels generally show little or no decline in activity between 150 and 80 Torr (see, for example, Ref. 20)], it is clearly less pertinent in the CNS, where ambient PO2 has consistently been reported to be below 40 Torr and may be as low as 20 Torr in the human brain (6). With this in mind, we recently studied the effect of a graded reduction in PO2 on resting and stimulated activity of recombinant human TREK1 (hTREK1) (18). Like its murine homologue (mTREK1), hTREK1 was robustly activated by arachidonic acid and membrane distortion by using chemical cup formers and crenators. However, reduction in PO2 to 60 Torr and below evoked sustained and significant channel inhibition. Most importantly, hypoxia completely reversed hTREK1 channel activation by either arachidonic acid or membrane stretch. Furthermore, when the order of modulator addition was reversed, hypoxia completely occluded any effect of arachidonic acid (18). These recent data emphasize two important points: first, hTREK1 is the latest addition to the steadily increasing array of O2-sensitive ion channels (see Refs. 8, 11, 21), and second, where such channels are expressed in the CNS, it is of paramount importance to study their regulation at physiologically pertinent PO2 levels (6).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. The full-length hTREK1 (KCNK2) was cloned and stably expressed in human embryonic kidney (HEK-293) cells as previously described (15, 18), using the vector pcDNA3.1/V5-His-TOPO containing a His6 and V5 epitope for immunocytochemical verification of protein expression (data not shown). No immunoreactivity or TREK-like currents were observed in untransfected cells (data not shown). Cells were maintained in Earle's minimal essential medium (containing L-glutamine) supplemented with 10% fetal calf serum, 1% antibiotic antimycotic, 1% nonessential amino acids, 0.2% gentamicin, and 0.1% geneticin G-418 sulfate (all purchased from GIBCO BRL, Paisley, Strathclyde, UK) in a humidified incubator gassed with 5% CO2-95% air. Cells were passaged every 7 days in a ratio of 1:25 with the use of Ca2+- and Mg2+-free phosphate-buffered saline (GIBCO BRL).

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 5–7 M{Omega}. 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Static modulation of intracellular pH: whole cell recording of hTREK1 currents. Upon transition from the cell-attached to the whole cell configuration (patch rupture), hTREK1 K+ currents steadily increased to reach a plateau at or within 2.4 min (Fig. 1A). Mean current densities immediately following patch rupture (Fig. 1B) were essentially independent of pipette pH [157 ± 46 pA/pF at pH 7.9 (n = 5), 173 ± 53 pA/pF at pH 7.2 (n = 13), and 282 ± 115 pA/pF at pH 6.5 (n = 6); P > 0.1, one-way ANOVA, Bonferroni post hoc test]. However, at each pipette pH value, currents significantly (P < 0.05, Student's paired t-test) ran up to plateau values of 263 ± 63 pA/pF at pH 7.9, 410 ± 80 pA/pF at pH 7.2, and 654 ± 198 pA/pF at pH 6.5 (Fig. 1B). Thus the final plateau value was dependent on intracellular pH, with the mean current densities demonstrating significant differences (P < 0.05, one-way ANOVA) at pH 7.9 and 6.5 (Bonferroni post hoc test, P < 0.05; Fig. 1B); at pH 7.2, mean current density ran up to an intermediate value (410 ± 80 pA/pF; Fig. 1B).



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Fig. 1. Effect of static changes in pipette pH on human tandem P domain K+ channel (hTREK1) whole cell currents. A: exemplar "run-up" profiles of whole cell currents after transition from cell-attached to whole cell configuration when pipette pH was buffer to the values indicated above each curve. Patch rupture occurred at t = 0 s. B: mean whole cell current densities at each pipette pH value indicated on the x-axis immediately after transition to whole cell configuration (Rupture; {bullet}) and after maximal current run-up had occurred (Plateau; {blacksquare}).

 

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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Fig. 2. Effect of dynamic changes in intracellular pH on hTREK1 whole cell currents. A: exemplar time courses of current modulation by intracellular acidification with bath application of 10 mM propionate or alkalinization with bath application of either 20 mM ammonium or 20 mM trimethylamine (TMA). Horizontal bar indicates periods of perfusion with weak acid and bases. B: mean, normalized current densities induced by dynamic modulation of intracellular pH using the agents indicated.

 

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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Fig. 3. Modulation of the acid response by hypoxia. A: typical whole cell hTREK1 currents evoked by ramp-step protocol during perfusion with agents indicated to right of current traces. Inset: whole cell voltage protocol, with associated pipette potentials, applies to Figs. 3, 4, 5, 6, 7, 8, 9, 10. B: exemplar time course of the effect of intracellular acidification with 10 mM propionate (horizontal bar) before and during bath hypoxia (hatched area) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 



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Fig. 4. Modulation of the alkaline response by hypoxia. A: typical whole cell hTREK1 currents evoked by ramp-step protocol during perfusion with agents indicated to right of current traces [TMA(#1) and (#2) indicate the first and second TMA-alone conditions, respectively]. B: exemplar time course of the effect of intracellular alkalinization with 20 mM TMA (horizontal bar) before and during bath hypoxia (hatched area) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 


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Fig. 5. Modulation of the hypoxic responses by acidosis. A: typical whole cell hTREK1 currents evoked by ramp-step protocol during perfusion with agents indicated to right of current traces (Prop, 10 mM propionate; H, hypoxia). B: exemplar time course of the effect of intracellular acidification with 10 mM propionate (horizontal bar) during bath hypoxia (hatched area) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 


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Fig. 6. Modulation of the hypoxic responses by alkalosis. A: typical whole cell hTREK1 currents evoked by ramp-step protocol during perfusion with agents indicated to right of current traces [Hypoxia(#1) and (#2) indicate the first and second hypoxia-alone conditions, respectively]. B: exemplar time course of the effect of intracellular alkalinization with 20 mM TMA (horizontal bar) during bath hypoxia (hatched areas) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 


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Fig. 7. Modulation of responses to acidosis and arachidonic acid by hypoxia. A: typical whole cell hTREK1 currents evoked by ramp-step protocol during perfusion with agents indicated to right of current traces (Prop, 10 mM propionate; H, hypoxia; AA, 10 µM arachidonic acid). B: exemplar time course of the effect of intracellular acidification with 10 mM propionate and arachidonic acid (horizontal bars) before and during bath hypoxia (hatched area) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 


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Fig. 8. Modulation of responses to alkalosis and arachidonic acid by hypoxia. A: typical whole cell hTREK1 currents evoked by ramp-step protocol during perfusion with agents indicated to right of current traces (TMA, 20 mM TMA; H, hypoxia; AA, 10 µM arachidonic acid). B: exemplar time course of the effect of intracellular alkalinization and arachidonic acid (horizontal bars) before and during bath hypoxia (hatched areas) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 


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Fig. 9. Modulation of responses to acidosis and hypoxia by arachidonic acid. A: typical whole cell hTREK1 currents evoked by ramp-step protocol during perfusion with agents indicated to right of current traces (Prop, 10 mM propionate; H, hypoxia; AA, 10 µM arachidonic acid). B: exemplar time course of the effect of intracellular acidification (with 10 mM propionate) and arachidonic acid (horizontal bars) during bath hypoxia (hatched area) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 


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Fig. 10. Modulation of responses to alkalosis and hypoxia by arachidonic acid. A: typical whole cell hTREK1 currents evoked by ramp-step protocol during perfusion with agents indicated to right of current traces (TMA, 20 mM TMA; H, hypoxia; AA, 10 µM arachidonic acid). B: exemplar time course of the effect of intracellular alkalinization and arachidonic acid (horizontal bars) during bath hypoxia (hatched areas) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 
In complete contrast to the effect of relative hypoxia on acid activation, reduction of bath PO2 during TMA inhibition did not affect the current suppression due to intracellular alkalinization (Fig. 4, A, B, and C). Indeed, relative hypoxia in the presence of TMA caused a further reduction in current. Thus 20 mM TMA evoked an inhibition of mean current density from 456 ± 50 to 223 ± 39 pA/pF (P < 0.005, n = 6) that was further reduced to 110 ± 20 pA/pF (P < 0.01, n = 6) by the coapplication of relative hypoxia; again, this effect was freely reversible upon wash. The normalized current density data are shown in Fig. 4C, which also plots the similar additional inhibitory effect of relative hypoxia when coapplied with ammonium and reinforces the notion that relative hypoxia is not able to reverse the inhibition induced by intracellular alkalinization. Thus 20 mM ammonium reduced current density from 272.6 ± 36.3 to 152 ± 16.5 pA/pF (P < 0.005, n = 6), and density was further reduced in the additional presence of relative hypoxia to 122.3 ± 13.7 pA/pF (P < 0.01, n = 6).

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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Fig. 11. Modulation of cell-attached responses to acidification and arachidonic acid by hypoxia. A: typical cell-attached hTREK1 currents evoked by ramp protocol during perfusion with agents indicated to right of current traces (Prop, 10 mM propionate; H, hypoxia; AA, 10 µM arachidonic acid). Inset: cell-attached voltage protocol, with associated pipette potentials, applies to Figs. 11, 12, 13, 14. B: exemplar time course of the effect of intracellular acidification (with 10 mM propionate) and arachidonic acid (horizontal bars) before and during bath hypoxia (hatched area) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 


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Fig. 12. Modulation of cell-attached responses to hypoxia by acidification and arachidonic acid. A: typical cell attached hTREK1 currents evoked by ramp protocol during perfusion with agents indicated to right of current traces (Prop, 20 mM TMA; H, hypoxia; AA, 10 µM arachidonic acid). B: exemplar time course of the effect of intracellular acidification and arachidonic acid (horizontal bars) during bath hypoxia (hatched areas) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 



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Fig. 13. Modulation of cell-attached responses to alkalinization and arachidonic acid by hypoxia. A, top: typical cell-attached hTREK1 currents evoked by ramp protocol during perfusion with agents indicated to right of current traces, with scale expanded to show effect of TMA. Bottom: cell-attached hTREK1 currents from same cell as at top but with scale extended to show AA activation (TMA, 20 mM TMA; H, hypoxia; AA, 10 µM arachidonic acid). B: exemplar time course of the effect of intracellular alkalinization with 20 mM TMA and arachidonic acid (horizontal bars) during bath hypoxia (hatched area) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 


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Fig. 14. Modulation of cell-attached responses to hypoxia by alkalinization and arachidonic acid. A: typical cell-attached hTREK1 currents evoked by ramp-step protocol during perfusion with agents indicated to right of current traces (TMA, 20 mM TMA; H, hypoxia; AA, 10 µM arachidonic acid). B: exemplar time course of the effect of intracellular alkalinization and arachidonic acid (horizontal bars) during bath hypoxia (hatched areas) on normalized hTREK1 currents. C: mean, normalized current densities under the conditions indicated.

 

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).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although many data are accumulating to address the structure-function relationships that underpin the regulation of the mTREK1 channel, equivalent information pertaining to the modulating influences of the hTREK1 homologue are only recently beginning to emerge. However, where parallel data are available, it appears that both homologues behave, for the most part, in a similar manner. For example, activities of both recombinant mTREK1 and hTREK1 are regulated by membrane distortion (18, 22, 23) and unsaturated fatty acids (14, 15, 18, 23). The present data serve to show that hTREK1 is activated by intracellular acidification in a manner comparable to that reported for mTREK1 (13). Acute modulation by reduced O2 availability has not been studied in mTREK1, but hTREK1 activity is acutely depressed by relative hypoxia in a PO2-dependent manner with maximal channel inhibition occurring at and below 60 Torr (18). Importantly, TREK1 regulation is polymodal, since many of the modulating influences interact. This is true of hypoxia and arachidonic acid or membrane distortion with hTREK1 (18) and intracellular pH, fatty acid modulation, heat sensitivity, and membrane distortion with mTREK1 (7, 12, 13). The present data extend our knowledge of this polymodal channel regulation, highlight a number of important new factors concerning hTREK1, and also indicate important differences between murine and human homologues of this tandem P domain K+ channel.

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.


    ACKNOWLEDGMENTS
 
GRANTS

This work was funded by The British Heart Foundation and The Wellcome Trust.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. J. Kemp, School of Biomedical Sciences, Worsley Bldg., Univ. of Leeds, Leeds LS1 9JT, UK (E-mail: p.z.kemp{at}leeds.ac.uk).

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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