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J Biol Chem, Vol. 274, Issue 38, 26691-26696, September 17, 1999

Mechano- or Acid Stimulation, Two Interactive Modes of Activation of the TREK-1 Potassium Channel^*

François Maingret, Amanda J. Patel, Florian Lesage, Michel Lazdunski, and Eric Honoré

From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UPR 411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TREK-1 is a member of the novel structural class of K⁺ channels with four transmembrane segments and two pore domains in tandem (1, 2). TREK-1 is opened by membrane stretch and arachidonic acid. It is also an important target for volatile anesthetics (2, 3). Here we show that internal acidification opens TREK-1. Indeed, lowering pH_i shifts the pressure-activation relationship toward positive values and leads to channel opening at atmospheric pressure. The pH_i-sensitive region in the carboxyl terminus of TREK-1 is the same that is critically involved in mechano-gating as well as arachidonic acid activation. A convergence, which is dependent on the carboxyl terminus, occurs between mechanical, fatty acids and acidic stimuli. Intracellular acidosis, which occurs during brain and heart ischemia, will induce TREK-1 opening with subsequent K⁺ efflux and hyperpolarization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The near completion of the sequencing of the nematode Caenorhabditis elegans genome recently identified more than 80 K⁺ channel genes divided into three major structural classes: (i) the inward rectifiers with two TMS¹ and a single P domain; (ii) the Shaker types with six TMS and a single P domain comprising the voltage-gated Kvs, the calcium-activated Slo, the calcium-regulated SK, the Eag/Erg, and the KQT channels; and (iii) the two P types with 4TMS being the largest structural class (about 50 genes) (4-6). Despite an overall similar 4TMS/2P structure, the sequence identity between these channels is very low (less than 30%) (5, 6).

The mammalian family of 4TMS/2P K⁺ channels comprises TWIK-1, TWIK-2, TASK-1, TASK-2, TREK-1, and TRAAK (1, 7-11). TWIK-1 and TWIK-2 are widely distributed and encode K⁺-selective channels with a characteristic weak inward rectification (8, 11). TASK-1 is found principally in the pancreas, placenta, lung, brain, and heart (9, 12, 13). TASK-1 lacks intrinsic voltage sensitivity and is thus a pure background K⁺-selective channel. Moreover, TASK-1 is extremely sensitive to variations of extracellular pH in a narrow physiological range, with 90% of the maximum current recorded at pH 7.7 and only 10% at pH 6.7 (9). TASK-2, another background K⁺ channel recently isolated from human kidney, shares the external pH sensitivity of TASK-1 (10). Unlike the other 4TMS/2P channels, TASK-2 is almost absent from the brain and is mainly expressed in the kidney. Murine TREK-1 is widely distributed with a strong expression in the brain and in the heart (1). It is activated by membrane stretch, by AA as well as inhalational anesthetics, while it is inhibited by a cAMP-dependent phosphorylation (2, 3). Interestingly, TREK-1 shares the properties of the Aplysia S-type K⁺ channel, which is involved in presynaptic facilitation underlying a simple form of learning (14, 15). TRAAK, another mouse mechano-gated AA-sensitive 4TMS/2P K⁺ channel, is only expressed in neuronal tissues including brain, spinal cord, and retina and lacks sensitivity to cAMP (7, 16).

The mammalian mechano-gated K⁺ channels that have been previously described in cardiac myocytes, neurons and epithelial kidney cells (17-30) share the biophysical and pharmacological properties of TREK-1, including single channel conductance (100 pS at 50 mV in symmetrical K⁺), flickery kinetics, voltage dependence, mechano-gating, and sensitivity to AA (2, 16).

In the present report, we demonstrate that the AA-sensitive mechano-gated K⁺ channel TREK-1 is opened by intracellular acidosis. Mutagenesis experiments identify the carboxyl-terminal region of TREK-1 as critical for the integration of both mechanical and acidic stimuli.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cDNA cloning, mutational strategy, cell culture, transfection, and electrophysiology procedures have been previously described elsewhere (2, 16). Briefly, murine TREK-1 and TRAAK cDNAs were cloned into pIRES-CD8 vector (1, 7). COS cells were transfected with the DEAE dextran procedure. The positive cells were visualized using the anti-CD8 antibody-coated bead method (2). Mutant TREK-1 46 was deleted at Thr-368, 89 at Thr-322, 100 at Arg-311, 103 at Gly-308, and 113 at Val-298. The chimera TRAAK/TREK-1 contained the core of TRAAK (truncated at Gly-255) and the carboxyl terminus of TREK-1 (Gly-293 to Thr-368).

For whole cell experiments, bath solution (EXT) contained 150 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, pH 7.4, with NaOH, and pipette solution (INT) contained 150 mM KCl, 3 mM MgCl₂, 5 mM EGTA, and 10 mM Hepes, pH 7.2, with KOH. The EXT K⁺-rich solution contained 150 mM KCl instead of 150 mM NaCl. The HCO₃ solution used to induce intracellular acidosis (31) was made by substituting 90 mM NaCl with 90 mM NaHCO₃. A K⁺-rich HCO₃ solution was made by substituting 90 mM KCl with 90 mM KHCO₃. For cell-attached experiments, the EXT solution contained 150 KCl instead of 150 NaCl, and the pipette contained the EXT solution (150 NaCl). To induce intracellular acidosis, 90 mM KHCO₃ was substituted for KCl (31). CO₂-rich solution was prepared by bubbling pure CO₂ in an EXT KCl solution (containing 25 mM HCO₃ instead of 10 mM Hepes) for 10 min (pH 6.0). The NH₄Cl prepulse EXT solution contained 20 mM NH₄Cl substituting for 20 mM KCl. For inside-out experiments, the pipette solution was EXT, and the bath solution was INT. For acidic (pH 5.0-6.0) INT solutions, Hepes was substituted with Mes, and for basic (pH 8.0) INT solution, Hepes was substituted with Tris. Hepes INT solutions at both acidic and basic pH gave similar results (not shown). Mechanical stimulation was applied through an open loop pressure generating system and monitored at the level of the patch pipette throughout the experiment by a calibrated pressure sensor. This system provides a stable pressure pulse (16). Pressure-effect relationships were fitted with Boltzmann equations. AA was dissolved in ethanol at a concentration of 100 mM, flushed with argon, and kept at 20 °C for 1 week. All chemicals were obtained from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TREK-1 cDNA was transiently transfected in COS cells, and channel activity was recorded using the whole cell patch clamp configuration. In a cell voltage-clamped at 0 mV, AA superfusion induces a strong outward current (Fig. 1A). In the same cell, repetitive application of 90 mM HCO₃, which produces intracellular acidification (31, 32), mimics AA stimulation (5.5 ± 0.5-fold increase, n = 29 at 0 mV). Both AA and HCO₃ are ineffective on control mock-transfected cells (0.5 ± 0.4, n = 7) (Fig. 1B). Moreover, substitution of 90 mM NaCl by an equivalent concentration of sodium gluconate does not mimic the activation of TREK-1 by NaHCO₃ (n = 6). The I-V curve of the current induced by HCO₃ shows a prominent outward going rectification in physiological K⁺ conditions and reverses at the predicted E_K⁺ value of 80 mV (Fig. 1C). When external Na⁺ is substituted with K⁺, the reversal potential shifts to 0 mV, and the I-V curve remains outwardly rectifying (Fig. 1D). AA similarly activates TRAAK, while HCO₃ is ineffective (0.9 ± 0.1, n = 17) (Fig. 1E).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Bicarbonate-mediated intracellular acidosis opens TREK-1 in the whole cell configuration. A, a COS cell expressing TREK-1 is studied in the whole cell configuration at a holding potential of 0 mV. Superfusion of 10 µM AA and 90 mM NaHCO3 (substituting NaCl) (as indicated by horizontal bars), which induces intracellular acidification, reversibly induces an outward current. Zero current is indicated by a horizontal dashed line. B, whole cell recording of a mock (CD8)-transfected COS cell (same conditions as A). C, I-V curve of the HCO3-induced current. The holding potential is 80 mV, and voltage ramps of 800 ms in duration are applied from 130 to 100 mV every 10 s. Both control and HCO3-rich solutions contain 5 mM K+. D, same cell as C in the presence of 155 mM K+. E, whole cell recording of a COS cell expressing TRAAK (same conditions as A).

The activation of TREK-1 by the addition of HCO₃ is also observed at the single channel level in the cell-attached patch configuration (n = 13) (Fig. 2A). In this configuration, the pH of the external solution bathing channels under recording is clamped by the pipette medium (pH 7.2), and channel modulation is expected to be due to intracellular effects. In this experiment, the activity of the mechano-gated channel TREK-1 is recorded both at atmospheric pressure and during the application of a 66 mm Hg pressure stimulation (Fig. 2A, inset). At atmospheric pressure, channel activity (NPo) is very low (0.80 ± 0.22, n = 27). Both the resting and the pressure-induced activities are reversibly stimulated by the HCO₃ addition (Fig. 2A). Superfusion of a CO₂-rich solution, which also produces a strong intracellular acidification (32), leads to TREK-1 opening in the cell-attached patch configuration (n = 15) (Fig. 2B). Again, both basal and stretch-induced activities are strongly stimulated. The current induced by CO₂ is outwardly rectifying and reverses at 80 mV (Fig. 2B, inset). Another classical approach to alter pH_i-regulated mechanisms is the NH₄Cl prepulse technique, which relies on the greater membrane permeability for NH₃ than for NH₄⁺ ions (32). The addition of NH₄Cl (producing intracellular alkalinization) does not affect TREK-1 channel activity, while washout of NH₄Cl (producing intracellular acidosis) strongly stimulates TREK-1 channel activity (n = 6) (Fig. 2C). The current activated by NH₄Cl withdrawal similarly displays a strong outward rectification (Fig. 2C).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Intracellular acidosis activates TREK-1 in the cell-attached patch configuration. A, cell-attached patch from a cell expressing TREK-1. Channel activity is recorded at atmospheric pressure and during a membrane stretch of 66 mm Hg (indicated by vertical arrows). The bath solution contains 155 mM KCl, and the holding potential is 0 mV. The application of 90 mM HCO3 (substituting KCl) in the bath is indicated by a horizontal bar. The inset shows currents induced by membrane stretch (66 mm Hg) in control and during HCO3 superfusion. Zero current is indicated by a horizontal dashed line. B, effects of a CO2-rich solution on a cell-attached patch from a cell expressing TREK-1 (same conditions as A). The inset shows I-V curves performed with voltage ramps of 800 ms in duration from 130 to 100 mV in control and during CO2 addition (different cell from B). C, effects of addition and washout of 20 mM NH4Cl (substituting KCl) on a cell-attached patch from a cell expressing TREK-1 (same conditions as A). During wash-out of NH4Cl, voltage is stepped to 50 and +50 mV (as indicated by arrows).

The effects of intracellular acidification were also studied on excised inside-out patches expressing TREK-1 (Fig. 3). Channel activity was recorded at both atmospheric pressure and during membrane stretch. Gradual intracellular acidification from 7.2 to 5.0 induces channel opening at atmospheric pressure (Fig. 3, A and B). NPo is strongly increased, while the single channel conductance is gradually decreased by internal acidosis (Fig. 3B). Half-maximal activation is induced at pH_i 6.0, and a drop of 0.7 pH_i unit already produces a significant increase in channel activity (Fig. 3B). A 42 mm Hg stretch induces a robust channel opening at intracellular pH between 7.2 and 6.0, although it fails to further open TREK-1 at pH_i 5.0 (Fig. 3A). The pressure-activity relationship of TREK-1 is presented in Fig. 3C. At physiological intracellular pH 7.2, the pressure-activity relationship is described by a Boltzmann function with a pressure required for half-maximal activity (P_0.5) of 36.8 ± 2.1 mm Hg (n = 13). Progressive lowering in pH_i gradually shifts the P_0.5 toward positive values, leading to constitutive channel activity under atmospheric pressure at pH_i 5.0 (Fig. 3, C and D). Half-maximal effect is observed at pH 5.9 (Fig. 3D).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Intracellular acidosis directly activates TREK-1 in the inside-out patch configuration. A, a membrane stretch of 42 mm Hg induces an outward current measured at 0 mV in an inside-out patch from a cell expressing TREK-1. Channel activity is recorded at various internal pH values (as indicated). Zero current is indicated by a horizontal dashed line. B, NPo measured at 0 mV under atmospheric pressure in inside-out patches expressing TREK-1. The number of patches is indicated. The effect of intracellular acidification on single channel current is illustrated in the inset. C, pressure-effect relationships constructed at different pHi values on the inside-out patch illustrated in A. Curves are fitted with Boltzmann equations, and P0.5 pressures are indicated by vertical arrows. D, intracellular acidification shifts TREK-1 half-maximal pressure activation (P0.5) toward positive values. The number of patches is indicated.

The modulation of channel activity by intracellular acidification was studied on deleted TREK-1 mutants in the inside-out patch configuration. We used a protocol including two acidic steps to pH_i 6.0 and 5.0 at both atmospheric pressure and during a 66 mm Hg stretch (Fig. 4, B-D). Lowering internal pH from 7.2 to 6.0 reversibly induces TREK-1 wild type opening at atmospheric pressure and slightly potentiates channel activity during stretch (Fig. 4B). Lowering internal pH to 5.0 further opens TREK-1 at atmospheric pressure with no additional effect at 66 mm Hg. Deletion of the N-terminal region of TREK-1 does not alter pH_i sensitivity or mechano-gating (n = 5; data not shown). However, serial deletion of the C terminus progressively impairs TREK-1 activation by pH_i (Fig. 4E). Like TREK-1 wild type, at atmospheric pressure, 46 is opened at pH 6.0 and 5.0 (Fig. 4E, second panel), while the next deletion inward, 89, lacks activation at pH 6.0 and is only mildly opened at pH 5.0 (Fig. 4, C and E, third panel). Activation of 89 by a membrane stretch of 66 mm Hg is strongly potentiated at both pH 6.0 and pH 5.0 (Fig. 4, C and E, third panel). 100 is only weakly activated at atmospheric pressure by pH 5.0, and channel activity induced by a 66 mm Hg stretch is only enhanced at pH 5.0 (Fig. 4E, fourth panel). 103 is not opened at atmospheric pressure by internal acidification at either pH 6.0 or pH 5.0, and stimulation of the stretch-induced activity is only observed at pH 5.0 (Fig. 4, D and E, fifth panel). No channel activity is detected with 113 at both pressures and at all pH conditions tested (n = 7).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   The carboxyl terminus of TREK-1 is critical for pHi activation. A, diagram illustrating the progressive deletions of the carboxyl terminus of TREK-1 (see "Experimental Procedures" for further details). B, inside-out patch from a cell expressing TREK-1 wild type recorded at a holding potential of 0 mV. Channel activity is recorded at atmospheric pressure and during a 66 mm Hg stretch (as indicated by vertical arrows). Applications of pH 6.0 and 5.0 in the internal solutions are indicated by horizontal bars. Zero current is indicated by a horizontal dashed line. C, same as B on an inside-out patch from a cell expressing the mutant 89. D, same as B on a cell expressing the mutant 103. E, histograms illustrating NPos measured at atmospheric pressure and during a 66 mm Hg stretch at various internal pH. The number of experiments are indicated. These measurements are performed in the inside-out configuration at a holding potential of 0 mV using the protocols illustrated in B, C, and D.

Progressive deletion of the cytosolic region of TREK-1 shifts the pressure-activity curve toward more negative values, leading to less sensitive mutant channels (Fig. 5, A-C). For instance, the relationship for 103 is about 60 mm Hg more negative compared with that of TREK-1 at pH 7.2 (Figs. 3C and 5C). Lowering pH_i to 6.0 and then 5.0 gradually shifts the relationship toward more positive values and strongly stimulates channel activity elicited by pressure (Figs. 4D and 5C).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   The carboxyl terminus of TREK-1 is critical for mechano-gating. A, mean pressure at 0 mV required for half-maximal activation (P0.5 value) of various TREK-1 mutants. P0.5 is determined by fitting Boltzmann equations to the pressure-effect relationships as illustrated in B and C. B and C, pressure-effect relationships for mutants 89 and 103. Pressure-effect relationship for TREK-1 wild type (WT) at pH 7.2 is indicated with a dashed line. P0.5 values are indicated by vertical arrows.

Unlike TREK-1, TRAAK, the other stretch- and AA-sensitive member of the 4TMS/2P K⁺ channel family (P_0.5 = 46 ± 2 at pH 7.2; n = 7) is quite insensitive to intracellular acidosis (Figs. 1E and 6, A and D). However, the high pH_i sensitivity of TREK-1 can be transferred to TRAAK when the proximal C-terminal regions are exchanged (Fig. 6, B, C, and E). As observed for TREK-1, internal acidosis shifts the pressure-activity curve toward positive values, resulting in the opening of the TRAAK/TREK-1 chimera at atmospheric pressure (P_0.5 = 40 ± 3, n = 7; P_0.5 = 32 ± 5, n = 4; P_0.5 = 26 ± 3, n = 4, at pH 7.2, 6.0, and 5.0, respectively). Moreover, HCO₃ superfusion induces a strong stimulation of the whole cell membrane current of the TRAAK/TREK-1 chimera (3.8 ± 0.4, n = 10), although it has no effect on TRAAK (0.9 ± 0.1, n = 17) (Fig. 1E).

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Transfer of proton sensitivity to TRAAK by fusing the proximal carboxyl terminus of TREK-1. A, intracellular acidification (as indicated by steps) does not alter TRAAK activity in an inside-out patch. The holding potential is 0 mV, and 66 mm Hg pressure applications are indicated by vertical arrows. Zero current is indicated by a horizontal dashed line. B, diagram illustrating the chimeric construct (see "Experimental Procedures" for further details). C, inside-out patch showing that internal acidosis stimulates TRAAK/TREK-1 chimera at both atmospheric pressure and during a stretch at 66 mm Hg (same conditions as A). D, histogram illustrating the effects of various pHi (as indicated) on TRAAK NPo recorded at atmospheric pressure and during a 66 mm Hg stretch. The number of experiments is indicated, and conditions are identical to A. E, same as D for chimera TRAAK/TREK-1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mammalian mechano-gated K⁺ channels have been previously described in atrial and ventricular cardiac myocytes, in neurons from mesencephalic and hypothalamic areas of the brain as well as in kidney (17-20, 29). Negative pressure applied to cell-attached patches activates K⁺ channels (17-20). The pressure to induce half-maximal activation is between 12 and 18 mm Hg at +40 mV. I-V curves are outwardly rectifying, and single channel conductances are 94 and 143 picosiemens at +60 mV in symmetrical K⁺ for cardiac and brain cells, respectively. Openings induced by stretch are typically bursty and flickery. The probability of these channels to open at a fixed pressure is voltage-dependent with a higher opening at depolarized potentials. Both cardiac and neuronal channels are similarly opened by AA and other lipophilic compounds in the micromolar range (17-20). AA activation is found in cells treated with cyclo-oxygenase and lipoxygenase inhibitors, indicating that AA itself can directly activate these channels. Unsaturated fatty acids (linoleic, linolenic, and docosahexaenoic acids) but not saturated fatty acids also activate these channels. A very important property of these native mechano-gated arachidonic-sensitive K⁺ channels is that they are also stimulated by lowering cytoplasmic pH over the range 7.2-5.6 (17, 20), and the channels are more sensitive to pressure at acidic intracellular pH.

Two recently cloned 4TMS/2P channels, TREK-1 and TRAAK (1, 2, 7, 16), have many of the properties of endogenous mechano-gated K⁺ channels. Both channels are activated by shear stress, cell swelling, and membrane stretch (2, 16). Moreover, TREK-1 and TRAAK are opened by AA, linoleic, linolenic, and docosahexaenoic acids but are resistant to saturated fatty acids (2, 7). The single channel conductance of about 100 pS at +50 mV in symmetrical K⁺, the outward going rectification, the bursty and flickery openings, and the voltage dependence are identical to described endogenous channels (2, 16-20).

The mechano-gated K⁺ channel TREK-1 is opened, like native cardiac and neuronal mechano-sensitive K⁺ channels (17-20), by intracellular acidosis. The mechanism by which pH_i variations activate TREK-1 involves a shift of the pressure-activity curve toward positive pressures, leading to constitutive channel opening at atmospheric pressure under mild internal acidosis. The acidic stimulation of TREK-1 was seen in whole cell (mediated by HCO₃), cell-attached (mediated by HCO₃, CO₂, and NH₄Cl prepulse), and excised inside-out patch configuration (internal acidification), suggesting that protons directly affect channel activity. To our knowledge, TREK-1 is the only cloned K⁺ channel reported so far to be directly opened by intracellular acidosis.

TRAAK is significantly (p < 0.05) less sensitive to stretch (P_0.5 = 46 ± 2 mm Hg) (16) compared with TREK-1 (P_0.5 = 36 ± 2 mm Hg). It is also much less sensitive to pH_i variations. This is a major difference between the two channels, which can now be used to ascribe a native AA- and mechanosensitive K⁺ channel to TREK-1 rather than to TRAAK. Another major difference between TREK-1 and TRAAK is the lack of inhibition by cAMP (7). Phosphorylation of Ser-333 in the carboxyl terminus of TREK-1 is responsible for the cAMP-induced down-modulation (2). The possibility that intracellular acidosis mediates TREK-1 opening via an interaction with the protein kinase A phosphorylation site, which is not present in the TRAAK structure, is ruled out, since 89, which lacks Ser-333 and is not sensitive to cAMP, remains stimulated by low pH_i.

Deletion and chimeric analysis indicate that the pH_i-sensitive region of TREK-1 is located in the carboxyl-terminal region between Val-298 and Thr-368 (113-46 region). Further deletion in this carboxyl-terminal region impairs activation by stretch, AA, and pH_i, demonstrating its critical importance for channel function (2). The sensitivity to pH_i is conferred to TRAAK when the proximal carboxyl terminus of TREK-1 is exchanged with TRAAK, demonstrating that the region between 46 and the fourth TMS is necessary and sufficient to provide pH sensitivity. Progressive deletions of the carboxyl terminus of TREK-1 show that pH_i sensitivity as well as mechano-gating is gradually altered. These results indicate that the whole segment (Val-298 to Thr-368) is probably involved in acidic and stretch modulation and that several amino acids may indeed be involved.

TREK-1 is modulated by a variety of mechanical stimuli such as stretch, swelling, and shear stress and by a variety of chemical stimuli including AA, ligands producing cAMP-dependent phosphorylation, and acidic stimuli (2, 3). TREK-1 is therefore an example of molecular integrator. Furthermore, mechanical activation of TREK-1 is enhanced by intracellular acidosis, demonstrating that a response to one type of stimulus alters the sensitivity to others.

By integrating multiple stimuli, TREK-1 probably fulfills an essential physiological function in the nervous and cardiovascular systems. Under physiological conditions, effectors of TREK-1 activity are probably stretch, AA, and neurotransmitters or hormones that increase intracellular cAMP (2). It is unlikely that activation of TREK-1 by acidification of the intracellular medium is an important physiological stimulus, although one cannot eliminate transient variations below pH_i 7.0 (33).

Protection against an exaggerated cellular Ca²⁺ invasion is one of the important roles of several types of K⁺ channels (34, 35). This has been particularly well established for the large conductance Ca²⁺-activated K⁺ channels (K_Ca²⁺ channels of the BK type) as well as for ATP-sensitive K⁺ channels (K_ATP channels) (36-38). When Ca²⁺ invades a cell, one of the ways to resist further Ca²⁺ invasion (which would be deleterious), from voltage-sensitive Ca²⁺ channels and/or through NMDA receptors, is hyperpolarization. Hyperpolarization puts the cell membrane potential far from the threshold of voltage-sensitive Ca²⁺ channel activation and favors the NMDA receptor-associated Ca²⁺-permeable channel blockade by Mg²⁺. It has been particularly well demonstrated that, when hypoxia, anoxia, or/and hypoglycemia occur, the associated decrease of intracellular ATP and the related increase of intracellular ADP result in the activation of K_ATP channels (34, 35, 39-41). This activation produced by inhibition of the energetic metabolism is protective in tissues where the channel is expressed such as in the heart or brain (35). In conditions of intracellular acidification, which would occur in many physiopathological situations such as brain or heart ischemia, opening of TREK-1 (K_Hi channel mode) may fulfill a similar protective role. TREK-1 channels in the K_Hi channel mode could work in concert with K_Ca²⁺ (BK type) and K_ATP channels. Cellular swelling, which also accompanies ischemia (42), would increase the effect of intracellular acidification on the activity of the TREK-1 channel (2). Ischemia in heart and brain is associated with a major K⁺ efflux (34, 35, 43). This efflux is supposed to be through K⁺ channels that open under these conditions (34, 35). However, specific blockers of K_Ca²⁺ channels or of K_ATP channels do not eliminate this efflux (43, 44). TREK-1 channels may thus constitute an important pathway for this K⁺ efflux.

	ACKNOWLEDGEMENTS

We are grateful to M. Jodar and D. Doume for excellent technical assistance.

	FOOTNOTES

^* This work was supported by the Center National de la Recherche Scientifique, the Association Française contre les Myopathies, the Conseil Régional Provence-Alpes Côte d'Azur, and the European Economic Community Marie-Curie Program (to A. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33 4 93 95 77 02/03; Fax: 33 4 93 95 77 04; E-mail: ipmc@ipmc.cnrs.fr.

	ABBREVIATIONS

The abbreviations used are: TMS, transmembrane segment(s); AA, arachidonic acid; Mes, 4-morpholineethanesulfonic acid; NPo, number of channels × open channel probability.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				R. M. Bryan Jr, B. K. Joseph, E. Lloyd, and N. J. Rusch Starring TREK-1: The Next Generation of Vascular K+ Channels Circ. Res., July 20, 2007; 101(2): 119 - 121. [Full Text] [PDF]

				M. Magra, S. Hughes, A. J. El Haj, and N. Maffulli VOCCs and TREK-1 ion channel expression in human tenocytes Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1053 - C1060. [Abstract] [Full Text] [PDF]

				A. Ohara, Y. Saeki, M. Nishikawa, Y. Yamamoto, and G. Yamamoto Single-channel Recordings of TREK-1 K+ Channels in Periodontal Ligament Fibroblasts. J. Dent. Res., July 1, 2006; 85(7): 664 - 669. [Abstract] [Full Text] [PDF]

				E. Honore, A. J. Patel, J. Chemin, T. Suchyna, and F. Sachs Desensitization of mechano-gated K2P channels PNAS, May 2, 2006; 103(18): 6859 - 6864. [Abstract] [Full Text] [PDF]

				A. Kloda, A. Ghazi, and B. Martinac C-Terminal Charged Cluster of MscL, RKKEE, Functions as a pH Sensor Biophys. J., March 15, 2006; 90(6): 1992 - 1998. [Abstract] [Full Text] [PDF]

				J. Murbartian, Q. Lei, J. J. Sando, and D. A. Bayliss Sequential Phosphorylation Mediates Receptor- and Kinase-induced Inhibition of TREK-1 Background Potassium Channels J. Biol. Chem., August 26, 2005; 280(34): 30175 - 30184. [Abstract] [Full Text] [PDF]

				J.-S. Kim, J.-Y. Park, H.-W. Kang, E.-J. Lee, H. Bang, and J.-H. Lee Zinc Activates TREK-2 Potassium Channel Activity J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 618 - 625. [Abstract] [Full Text] [PDF]

				D. Kang, C. Choe, and D. Kim Thermosensitivity of the two-pore domain K+ channels TREK-2 and TRAAK J. Physiol., April 1, 2005; 564(1): 103 - 116. [Abstract] [Full Text] [PDF]

				J. Chemin, A. Patel, F. Duprat, M. Zanzouri, M. Lazdunski, and E. Honore Lysophosphatidic Acid-operated K+ Channels J. Biol. Chem., February 11, 2005; 280(6): 4415 - 4421. [Abstract] [Full Text] [PDF]