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1G
Address correspondence to Karel Talavera Pérez, Laboratorium voor Fysiologie, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Fax: 32-16-34-59-91; email: karel.talavera{at}med.kuleuven.ac.be
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ABSTRACT |
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1G heterologously expressed in HEK-293 cells. AA inhibited 1G currents within a few minutes, regardless of preceding exposure to inhibitors of AA metabolism (ETYA and 17-ODYA). Current inhibition was also observed in cell-free inside-out patches, indicating a membrane-delimited interaction of AA with the channel. AA action was consistent with a decrease of the open probability without changes in the size of unitary currents. AA shifted the inactivation curve to more negative potentials, increased the speed of macroscopic inactivation, and decreased the extent of recovery from inactivation at –80 mV but not at –110 mV. AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern. We observed a tonic current inhibition, regardless of whether the channels were held in resting or inactivated states during AA perfusion, suggesting a state-independent interaction with the channel. Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve. Slow-inactivating 1G mutants showed an increased affinity for AA with respect to the wild type, indicating that the structural determinants of fast inactivation are involved in the AA–channel interaction.
Key Words: Low voltage–activated calcium channel • fatty acid • kinetic model • inactivation • mutant
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The activity of T-type channels may be affected by multiple neurotransmitters and hormones, extracellular pH, redox agents, and antihypertensive, anesthetic, antiarrythmic, and antiepileptic drugs (Vassort and Alvarez, 1994; Huguenard, 1996; Todorovic et al., 2001; Perez-Reyes, 2003).
Remarkably, it has been shown that two related endogenous compounds, arachidonic acid (AA) (Chesnoy-Marchais and Fritsch, 1994; Schmitt and Meves, 1995; Zhang et al., 2000) and the endocannabinoid anandamide (N-arachidonoylethanolamine [AEA]) (Chemin et al., 2001) modulate T-type channels. Both decrease the time constant of macroscopic inactivation and shift the steady-state inactivation curve to more negative potentials, without striking changes in the activation properties. Based on these data, Zhang et al. (2000) proposed that AA inhibits the T-type channel 1H by acting mainly on a voltage-dependent transition between closed states. However, the support of this hypothesis is quite weak for two reasons. First, in the framework of recent models for T-type channel gating (Serrano et al., 1999; Burgess et al., 2002), the putative effects of AA on voltage-dependent transitions between closed states have to be very pronounced to account for the large decrease in current amplitude, but these would modify the activation curve to a much larger extent than observed experimentally. Second, the characterization of the effects of AA on kinetic properties is still too incomplete to propose a proper model for modulation.
In the present study we examined the mechanism of inhibition of the T-type Ca2+ channel subunit 1G by AA in HEK-293 cells. We investigated whether AA effects were dependent on AA metabolism and performed a detailed characterization of changes induced in the gating kinetics of the channel. In addition, structural determinants of modulation by AA were probed by comparing the effects of AA on slow-inactivating mutants with those on the wild-type channel. Experimental data and model simulations suggest that current inhibition in physiological conditions results from the combination of two factors: the switch of the channels into a nonavailable conformation and the shift of the voltage dependence of the steady-state inactivation, which results from the alteration of transitions between inactivated states.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1G were investigated with whole-cell recordings using an extracellular solution containing (in mM) 2 CaCl2, 150 NaCl, 5 CsCl, 5 glucose, 1 MgCl2, and 10 HEPES and titrated to 7.4 with HCl. For the comparison of the effects on 1G and slow inactivating mutants and the test for tonic inhibition by AA we used 20 mM CaCl2 and 120 mM NMDG+ instead of NaCl. The intracellular solution contained 102 mM CsCl, 10 mM HEPES, 5 mM MgCl2, 5 mM Na2-ATP, 10 mM TEA-Cl, 10 mM EGTA, titrated to pH 7.4 with 1 N CsOH. For inside-out experiments, the pipette solution contained (in mM) 110 CaCl2, 10 HEPES, pH 7.4, and the bath solution 150 CsCl, 1 MgCl2, 1 Na2ATP, pH 7.2. AA, used in its Na-salt form, was dissolved in water. 5,8,11,14-Eicosatetraenoic acid (ETYA) and 17-octadecanoic acid (17-ODYA) were dissolved in DMSO. All chemicals were purchased from Sigma-Aldrich.
Electrophysiology and Data Analysis
Human embryonic kidney cells (HEK-293) were grown in Dulbecco's modified Eagles medium containing 10% (vol/vol) human serum, 2 mM L-glutamine, 2 U/ml penicillin, and 2 mg/ml streptomycin at 37°C in a humidity controlled incubator with 10% CO2. Transient transfection with the wild-type 1G or different constructs (Marksteiner et al., 2001; Staes et al., 2001) was performed using Trans-IT-293 reagents (Mirus). Currents were recorded using an EPC-7 (LIST Electronics) patch-clamp amplifier and filtered with an eight-pole Bessel-filter (Kemo). For control of voltage-clamp protocols and data acquisition, we used an IBM-compatible PC with TL-1 DMA or Digidata 1322A interfaces (Axon Instruments) and pCLAMP-software (Axon Instruments). Patch pipettes (1–2.5 M
) were pulled from Vitrex capillary tubes (Modulohm) using a DMZ-Universal puller (Zeitz Instruments). Series resistance was compensated to the maximum extent possible (40–60%). An Ag-AgCl wire was used as reference electrode. Membrane capacitive transients were electronically compensated and the linear background components were digitally subtracted when possible before data analysis. In the case of tail current measurements, linear background components and capacitive transients were subtracted by the application of a –P/4 protocol. Current traces were filtered at 2–5 kHz and digitized at 5–10 kHz. All experiments were performed at 22–25°C.
The activation curve was determined for each cell and experimental condition by dividing the I/V curve by a function Itail = Itail(V) that describes the open pore conduction (see below). Steady-state inactivation curves were fit by
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,b). In whole-cell recordings all voltage protocols, the holding potential was –110 mV except when stated otherwise. The stimulation frequency was adjusted for each particular protocol as to ensure at least 5 s of recovery at –110 mV. In inside-out experiments, the holding potential was –80 mV and the stimulation frequency 0.5 Hz.
The data were analyzed using WinASCD (G. Droogmans, KU Leuven; ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/winascd.zip). For all measurements, pooled data are given as mean ± SEM. We used Student's t test, taking P < 0.05 or P < 0.01 as level of significance. We used MATLAB (MathWorks) to solve and fit a Markov model for the gating of 1G in control and in the presence of 3 µM AA (Fig. 10). Parameter optimization and numerical solution of the differential equations were performed with the built-in functions fmin and expm, respectively. Voltage-dependent rate constants were written in the form
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i account for the coupling between the local electric potential sensed by qi and the membrane potential V. T = 25.4 mV is the thermal energy in electron volts. |
RESULTS |
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1G currents evoked by depolarizations from –110 to –20 mV to 54 ± 4% (n = 6) of the control value and decreased the time constant of inactivation within a few minutes (Fig. 1 A). Inhibition of 1G current was dose dependent, with an effective concentration (EC50) of 3.9 ± 0.3 µM and a Hill coefficient (N) of 1.6 ± 0.2 (Fig. 1 B).
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1G1G current (99 ± 1 of the control value; n = 5) or the inhibition by AA (2 µM; 21 ± 4% in control [n = 3] and 21 ± 5% in the presence of 17-ODYA [n = 5]; Fig. 2 B).
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1G in cell-free inside-out patches with similar potency and time course to those observed in whole-cell experiments. Fig. 3 A shows an example of the AA-induced inhibition of currents evoked by voltage ramps from –80 to +20 mV in an inside-out patch. AA effect was rapidly reversed upon washout using BSA (Fig. 3, A and B). In average, application of 3 µM AA induced a 52 ± 4% peak current inhibition (n = 5). Fig. 3 C exemplifies the inhibitory effect of AA on currents evoked by depolarizing pulses from –80 to –10 mV. AA decreased the open probability and promotes the occurrence of null sweeps. Current inhibition can be confirmed by the comparison of the average of current traces recorded in control and in the presence of AA (Fig. 3 D). Notably, both the extent of current inhibition and the acceleration of the macroscopic inactivation were similar to those observed in whole-cell experiments (see Fig. 1 and Fig. 4, B and E). In summary, these results indicate that the inhibition of 1G by AA is not mediated by any of its metabolites and occurs in a membrane-delimited manner.
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Arachidonic Acid Effects on the Gating of 1G
To study the effects of AA on the gating properties of 1G we compared whole-cell data obtained in control conditions with those collected at the steady-state level of inhibition after a 3–4-min perfusion with 3 µM extracellular AA.
Activation and Inactivation
In order to study the effects of AA on activation properties of 1G, current–voltage relationships (I/V curves) were determined from the peak amplitude of currents elicited by 200 ms–lasting voltage steps from a holding potential of –110 mV to potentials varying from –80 to 60 mV (Fig. 4 B, inset). Fig. 3 A exemplifies the effects of AA on the current traces elicited by steps to –60, –45, –30, or 0 mV. As can be observed in the I/V curves (Fig. 4 B), AA induced a significant decrease of the current amplitude at potentials positive to –55 mV. Fig. 4 C shows that AA did not significantly modify the activation curve at potentials positive to –55 mV and caused a small but significant increase of activation near the threshold (inset in the right). Steady-state inactivation curves h
= h(V) were determined from normalized peak amplitudes of currents recorded at –20 mV after 5 s–lasting prepulses to potentials from –110 to –55 mV (Fig. 4 C, left inset). AA shifted the inactivation curve to more negative potentials, with a voltage for half-maximal inactivation Vinac = –87.3 ± 1.0 mV compared with –74.2 ± 0.8 mV in control (Fig. 4 C). However, it did not affect the voltage sensitivity of steady-state inactivation (sinac = 4.3 ± 0.2 mV in control and sinac = 4.35 ± 0.09 mV in the presence of AA).
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inac). AA significantly decreased tp at potentials negative to –55 mV, but did not significantly alter the values at depolarized potentials (Fig. 4 D). On the other hand, inac was significantly reduced at all voltages (Fig. 4 E).
Kinetics of Inactivation from Closed States
The results described above indicate that AA speeds up the transition from the open to the inactivated state, but give no clues about the possible effect of AA on the rate of inactivation from closed states. We therefore studied the effects of AA on the time course of the onset of inactivation at potentials below the threshold of channel activation. This was achieved by measuring the amplitude of the currents elicited by pulses to –20 mV, applied after steps of variable duration to –85 or –75 mV (Fig. 5 C, inset). These amplitudes were normalized to that of the current elicited with a pulse to –20 mV from a holding potential of –110 mV. The data were then subtracted from 1 and plotted as a function of the time of sojourn at –85 or –75 mV. Fig. 5 shows that AA increased the steady-state level of inactivation at both –85 and –75 mV, in accordance with the hyperpolarizing shift of the inactivation curve. However, AA only slightly increased the rate of inactivation from closed states, as shown by the comparison of normalized data (Fig. 5 D).
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(V) with parameters Vreac = –76.9 ± 0.7 mV and sreac = 3.4 ± 0.2 mV. AA shifted the voltage dependence of channel recovery to more negative potentials (Vreac = –89.3 ± 0.1 mV) and decreased the voltage sensitivity (sreac = 4.02 ± 0.09 mV).
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decay) from a single-exponential fit of the time course of the tail currents. decay corresponds to the time constant of deactivation (deac) at potentials negative to the activation threshold and to the time constant of the macroscopic inactivation (inac) at supra-threshold potentials. Fig. 7 A shows that AA does not affect the voltage dependency of the time constant of deactivation of 1G (deac). On the other hand, AA decreased the time constant of tail current decay at potentials positive to the activation threshold, accordingly with the effects observed for the time constant of macroscopic inactivation (Fig. 4 E). Application of 3 µM AA decreased the amplitude of tail currents to 
50% without significant effects on the rectification pattern or the reversal potential of the channels (Fig. 7 B). This indicates that AA does not interact with the open state of the channel so as to affect its macroscopic conduction properties.
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1Gonset = 14 ± 2 s, which is smaller than that for the inhibition of the current evoked from –110 mV (inh = 57 ± 2 s), indicating that the changes of current amplitude and of the rate of inactivation from closed states are temporally distinct (see inset). In contrast, the time course of the rate of inactivation from the open state (inac = 37 ± 2 ms; Fig. 8 C) correlates well with that of the reduction in current amplitude.
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1G1G is state dependent, we used the following protocol. First, current amplitude was monitored in control by repetitive depolarizations from –100 to 0 mV. Stimulation was then stopped and cells were incubated with 3 µM AA for 3 min, during which the membrane potential was clamped at either –100 or 0 mV ([Ca2+]ext = 20 mM). Stimulation was then resumed with the same protocol used in control. Tonic inhibition was estimated from the comparison of the peak current in control and the peak current in the first pulse after resuming stimulation. We observed a tonic inhibition of 32 ± 5% (n = 5) and 39 ± 4% (n = 3) for perfusion periods at –100 and 0 mV, respectively, and no further significant decrease of current amplitude. The values of tonic inhibition were not significantly different to the level of steady-state inhibition by AA during repetitive stimulations (37 ± 5%; n = 8).
Molecular Determinants of Macroscopic Inactivation of 1G Modulate the Inhibition by Arachidonic Acid
The results described above indicate that AA modifies the inactivation of 1G. This suggested to us that 1G mutants that show slowed macroscopic inactivation (Marksteiner et al., 2001; Staes et al., 2001) might display different sensitivity to AA. Fig. 9 A shows a representation of the structure of 1G and the mutants investigated. GC
C4 is a chimera in which the last 409 amino acids of 1G are replaced by the amino acids 1540–2166 of 1C. This chimera conserves the first 23 amino acids of the COOH terminus we have previously reported to be necessary for fast inactivation of 1G (Staes et al., 2001). In GCC5 and GC1, the first 23 amino acids of the COOH terminus of 1G were substituted by the corresponding stretch of 1C or deleted, respectively. Marksteiner et al. (2001) described what they called a "hot spot" of determinants of fast inactivation in the S6 segment of the third domain of 1G. To diversify the structural determinants of fast inactivation to be tested for AA sensitivity, we also studied the mutant M1510I, which shows an 10-fold decrease in the rate of macroscopic inactivation with respect to 1G.
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1G and the slow-inactivating mutants GCC5, GC1, and M1510I by 3 µM AA. All these mutants were inhibited faster and to a larger extent than 1G (Fig. 9, C and E). The higher sensitivity of slow-inactivated mutants was confirmed by the comparison of the dose–response curves (Fig. 9 D). The values of EC50 and Hill coefficient (N) for GCC5, GC1, and M1510I were significantly lower than for the wild type (Fig. 9 F). Notably, the GCC4 mutant, which inactivates as fast as 1G (Staes et al., 2001), showed a nearly identical sensitivity to that of the wild type. Taken together, these data suggest that the AA sensitivity is modulated by the structural determinants of fast inactivation of 1G.
A typical feature of AA modulation of T-type channels is the decrease of the time constant of inactivation. Remarkably, 1G and the GCC4 mutant showed similar reduction of this parameter with increasing doses of AA (Fig. 9 G). The effective concentration and the Hill coefficient describing the pooled data of both channels were 2.4 ± 1.2 µM and 1.5 ± 0.8, respectively. In contrast, AA did not affect the time constant of inactivation of any of the slow-inactivating mutants (Fig. 9 G), indicating that the increase of the rate of inactivation from the open state is not a determinant for AA-induced inhibition.
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DISCUSSION |
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1G by arachidonic acid as an essential step in understanding its influence on the physiology of T-type channels. To our knowledge, this is the first study applying model simulations to provide for a mechanism of AA modulation of the gating of a voltage-dependent channel and to address directly the structural determinants of AA interaction with voltage-dependent Ca2+ channels. We demonstrate that AA can inhibit 1G currents directly, by exerting two independent effects, (a) reducing channel availability and (b) shifting the voltage dependence of the steady-state inactivation to more negative potentials. We also show that AA interacts with 1G in both resting and inactivated states and that structural determinants of inactivation modulate the affinity for AA.
We show that AA effects on 1G are 10-fold faster and more potent than those reported for 1H (Zhang et al., 2000). This difference is probably due to an 1 subunit specificity of AA effects. Schmitt and Meves (1995) reported that in the NG108-15 cell line, which mainly expresses 1H (Chemin et al., 2002), AA produced a continuous and slow decrease of T-type current amplitude with a similar range of potency (25–100 µM) to that observed by Zhang et al. (2000). In contrast, Chesnoy-Marchais and Fritsch (1994) showed that the effects of 2–10 µM AA on the T-type currents of rat osteoblastic cells, which mainly express 1G (Gu et al., 1999), developed within a few minutes, more like the results presented here.
AA Interacts with 1G in a Membrane-delimited Fashion
To fully understand the mechanism of AA inhibition it is important to know whether AA and/or some of its metabolites or PKC activation are responsible for this effect. Zhang et al. (2000) showed that the inhibition of the T-type channel subunit 1H is partially antagonized by the preincubation with the epoxygenase inhibitor 17-ODYA or by the intracellular perfusion of a PKC inhibitor. However, Park et al. (2003) reported the absence of inhibitory effects of PKC in the three T-type isoforms when expressed in HEK-293 cells. We confirmed this result for 1G (unpublished data). Our data indicate that AA is directly responsible for the decrease of the 1G current, as the application of 17-ODYA and ETYA, a nonmetabolizable analogue and inhibitor of the AA metabolism, did not influence the inhibition by AA (see also Schmitt and Meves, 1995). It could be also speculated that AA acts through its conversion to anandamide via the reverse mode of fatty acid amide hydrolase (FAAH). However, this hypothesis can be rejected as AA has stronger effects on 1G than anandamide (Chemin et al., 2001; see below). Furthermore, AA was able to inhibit 1G in cell-free inside-out patches with similar potency and time course to those observed in whole-cell experiments. In addition, FAAH is not expected to produce anandamide from AA in inside-out patches given the lack of ethanolamine in the bath solutions (Arreaza et al., 1997). In summary, we demonstrated that AA inhibits 1G channels in a membrane-delimited manner, though it remains to be elucidated whether this action is exerted by binding to the protein itself or, indirectly, by modifying the lipid environment.
Modulation of 1G Gating by AA: Mechanistic Insights from Kinetic Modeling
Currently available information on the effects of AA on the gating properties of T-type channels is scarce and does not provide solid support to previous hypotheses on the mechanism of modulation (see INTRODUCTION). We therefore studied the effect of AA on the voltage dependencies of activation and steady-state inactivation, the rate of inactivation from open and closed states, the recovery from inactivation, the deactivation, and on the macroscopic conduction properties of the T-type channel 1G. To analyze these data in a compact and consistent way we used a Markov model that describes the gating properties of 1G (Fig. 10), following a similar approach to that previously used to study the effects of extracellular pH and Ca2+ concentration (Talavera et al., 2003a) and mutations in the selectivity filter (Talavera et al., 2003b) on the gating properties of 1G. Table I lists the optimal values of model parameters that describe the data in control and in the presence of 3 µM AA (Figs. 4– 7). The interpretation of these results is given in the following paragraphs.
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1–3) did not improve the fit of the data obtained in AA. AA shifts the foot of the activation curve of 1G to more negative potentials (Fig. 4 C). This can be accounted for by varying the rate constant of the transitions between the closed states of the activation pathway, especially by decreasing KC3C2 (Table I and Fig. 10). Interestingly, this result may also explain the observations of Zhang et al. (2000), who described the effects on the activation curve of the T-type channel 1H as an increase of the slope factor. More dramatic shifts in the activation curve of T-type channels were described previously (Chesnoy-Marchais and Fritsch, 1994; Schmitt and Meves, 1995), and also for high voltage–activated Ca2+ channels (Barrett et al., 2001; Liu et al., 2001) and Na+ channels (Lee et al., 2002). On the other hand, AA had no significant effect on the deactivation properties of 1G (Fig. 7), indicating that it does not affect the transition from the open to the nearest closed state (O 
C3; Fig. 10).
In contrast, AA profoundly modified the inactivation properties by shifting the voltage dependence of steady-state inactivation by 13 mV to more negative potentials (Fig. 4 C), an effect that was reflected in the voltage dependence of the recovery from inactivation (Fig. 6 E). However, AA only slightly increased the rate of inactivation from closed states (onset of inactivation) and decreased the rate of recovery from inactivation at –80 mV (Fig. 5 D and inset of Fig. 6 C). These results can be explained by a slight increase of rate of the transition I1 I2 and a more pronounced increase of the rate of transition I2 I3, which were partially balanced by a decrease of the rate of the transition I3 IO (Table I and Fig. 10). We propose that the increase in the rate of transitions between inactivated states in the forward (activation) direction is the basis of the negative shift of the inactivation curve induced by AA and related compounds in voltage-dependent Na+ (Fraser et al., 1993; Xiao et al., 1995; Vreugdenhil et al., 1996; Bendahhou et al., 1997; Lee et al., 1999, 2002; Leifert et al., 1999) and Ca2+ channels (Chesnoy-Marchais and Fritsch, 1994; Schmitt and Meves, 1995; Petit-Jacques and Hartzell, 1996; Unno et al., 1996; Zhang et al., 2000; Chemin et al., 2001).
We also observed a decrease of the time constant of macroscopic inactivation (inac), but in contrast with Zhang et al. (2000), AA did not affect the voltage sensitivity of this parameter but induced a more uniform reduction over the whole voltage range. This is accounted for by an 14% increase in the rate of the transition between the open state and the nearest inactivated state (O IO; Table I).
One of the most important conclusions that can be derived from the experimental data and the kinetic modeling is that the decrease in current amplitudes for depolarizations from a holding potential of –110 mV (Fig. 4 B) cannot be explained by any of the AA effects on macroscopic gating we observed. Our results demonstrate that this decrease does not result from the modification of the conduction properties of 1G since AA did not change the unitary current and did not significantly modify the rectification pattern of the open pore conduction or the reversal potential of 1G. Notably, AA did not affect the unitary conductance of Ca2+ (Liu and Rittenhouse, 2000), Na+ (Xiao et al., 1995; Bendahhou et al., 1997), Cl– (Anderson and Welsh, 1990; Hwang et al., 1990), K+ (Bringmann et al., 1998; Devor and Frizzell, 1998), and NMDA receptor channels (Miller et al., 1992).
Single channel data from L-type Ca2+ channels of rat sympathetic neurons revealed that the AA-induced reduction of macroscopic current is due to an increased probability of null sweeps (Liu and Rittenhouse, 2000). Our results from inside-out patches are consistent with this mechanism (Fig. 3 B). Within this view, AA induces a fraction of channels to transit to a nonfunctional conformation or, alternatively, an increase in the rate of inactivation from closed states (Fig. 4 B and Table I). We denote, therefore, that AA exerts two different actions on the gating of 1G, one on the transitions between inactivated states and another by which it decreases the fraction of channels that are available to open. The distinct time courses of the decrease of current amplitude and the increased inactivation from closed states (Fig. 8) provide further support for this contention. In addition, this observation indicates that the interaction of AA with the channel is in the range of several seconds, in contrast with the estimate of minutes obtained from the decrease of current amplitude.
We found that AA induced similar tonic inhibition after perfusion periods during which the membrane potential was held at –100 or 0 mV. This proves that AA interacts indistinctively with both the resting and inactivated states of 1G. The analysis of the data by model simulations indicates, however, that this interaction only alters certain gating transitions.
Molecular Determinants of Macroscopic Inactivation Modulate AA Sensitivity
AA effects on the inactivation properties of 1G suggested us to test the effects of AA on several slow-inactivating mutants of 1G (Fig. 9 A) (Marksteiner et al., 2001; Staes et al., 2001). Remarkably, all of them showed an enhanced AA sensitivity with respect to 1G. Furthermore, the GCC4 chimera, with a major modification in the COOH terminus but the same fast-inactivation pattern as the wild-type 1G (Staes et al., 2001), showed a similar sensitivity to that of 1G. To explain these results, it could be envisaged that AA inhibits preferentially noninactivated channel conformations. However, this is contradicted by the absence of inhibition of wild-type currents at potentials at which there is a relatively slow rate of inactivation (i.e., close to the activation threshold; Fig. 4 B) and by the tonic inhibition observed after AA was applied at 0 mV (channels in the inactivated state). The simplest explanation for these observations is that the mutations we studied induce an increase in the affinity of a putative AA–channel interaction, independently of the effects on the rate of macroscopic inactivation.
Further analysis of the data obtained for the mutant channels allows us to extract some complementary conclusions. First, the AA–channel interaction is not determined by the specific sequence of the last 409 amino acids of the COOH terminus of 1G. Second, AA does not significantly inhibit 1G by acting on the open state, since it does not modify the rate of inactivation from the open state in the slow-inactivating mutants, in contrast to the case of 1G and the GCC4 chimera. And third, the faster inhibition observed for the slow-inactivating mutants (Fig. 9 C) indicates that channel conformation is an important determinant of the rate of inhibition, in contrast with the hypothesis of slow metabolism of AA to other active compounds as the only cause of the slow action of AA (Zhang et al., 2000).
Interestingly, the structural determinants of modulation by AA or analogue compounds have been also mapped in S6 transmembrane segments in the human cardiac Na+ channel hH1 (Xiao et al., 2001) and the intermediate conductance Ca2+-activated K+ channel hIK1 (Hamilton et al., 2003) or in intracellular domains, NH2 terminus of ROMK1 (Macica et al., 1998), and COOH terminus of TREK-2 (Kim et al., 2001), TREK-1 (Patel et al., 1998; Maingret et al., 2000), and Kir3.4 (Rogalski and Chavkin, 2001). Given the low sequence analogy among these channels, including T-type channel 1G, it is likely that fatty acid–channel interactions are determined by analogous tertiary structures rather than by the presence of conserved consensus binding motifs.
Functional Relevance of AA Modulation of T-type Channels
AA is a ubiquitous second messenger that modulates a wide range of cellular processes (Piomelli, 1993). We have shown that AA directly inhibits the opening of 1G, which limits the contribution of this channel to the early depolarization phase in excitable tissues. In addition, AA significantly shifts the inactivation curve to more negative potentials without dramatic effects on the activation curve. Consequently, AA is expected to decrease the window current, thereby limiting the Ca2+ influx through 1G at basal membrane potentials in both nonexcitable and excitable tissues. Therefore, 1G is a possible target for AA-induced cytoprotection and decrease of neuronal and cardiac excitability. Indeed, in parallel to the action of some antiepileptic drugs (Huguenard, 1996; Heady et al., 2001), the inhibition of T-type currents due to accumulation of free AA (Bazan, 1989; Van der Vusse et al., 1997) may underlie a mechanism of protection against neuronal and cardiac damage during epileptic and ischemic episodes. Analogously, the antiarrhythmic (Kang and Leaf, 2000), bradycardic (Mullane et al., 1979), and hypotensive (Damas and Troquet, 1978; Lee and Murthy, 1979; Mullane et al., 1979) effects of AA may be partially due to the inhibition of T-type channels, but further investigation is required to support these hypotheses.
Chemin et al. (2001) reported the inhibition of recombinant and native T-type channels by the endocannabinoid anandamide (AEA) and discussed the possible significance of this modulation in the mechanisms of slow-wave sleep, epilepsy, nociception, heart pacemaking, and cellular proliferation and development. These authors argued for a direct effect of AEA on T-type channels. However, we show here that the product of AEA hydrolysis via the action of FAAH, AA, also has an inhibitory effect on 1G. In fact, in addition to the negative shift of the inactivation curve, AA inhibits this channel at very negative holding potentials, in contrast to the observations with AEA. Chemin et al. (2001) showed that 3 µM AEA produces 40% inhibition of 1G current amplitude at a holding potential of –80 mV, while we predict that, at the same concentration, AA induces a 90% inhibition at this holding potential. This indicates that after AEA hydrolysis, the effect on 1G is enhanced through the action of AA. In contrast, comparison with studies on 1H (Schmitt and Meves, 1995; Zhang et al., 2000) suggests that hydrolytic function of FAAH attenuates the effects of AEA, due to the relatively lower sensitivity of this channel to AA.
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ACKNOWLEDGMENTS |
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1G clone (Technische Universität München, München, Germany). We are also grateful to Drs. T. Voets, J.L. Alvarez, G. Vassort, G. Owsianik, K. Mubagwa, and J. Vereecke for helpful discussions. The expert technical assistance of J. Prenen, M. Crabbé, H. Van Weijenbergh, S. de Swaef, and M. Schuermans is greatly acknowledged. This work was supported by the Belgian Federal Government (Interuniversity Poles of Attraction Program, Prime Ministers Office, IUAP Nr.3P4/23), the Flemish Government, and the Onderzoeksraad KU Leuven (GOA 99/07, F.W.O. G.0237.95, F.W.O. G.0214.99, F.W.O. G.0136.00).
Olaf S. Andersen served as editor.
Submitted: 1 March 2004
Accepted: 8 July 2004
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REFERENCES |
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1H calcium channels induce neuritogenesis and expression of high-voltage-activated calcium channels in the NG108-15 cell line. J. Neurosci. 22:6856–6862.1C, 1D and 1G subunits. Pflugers Arch. 438:553–560.[CrossRef][Medline]1G T-type calcium channel. J. Gen. Physiol. 114:185–201.1G. J. Physiol. 530:35–45.1G (Cav3.1). J. Gen. Physiol. 121:511–528.1G. J. Gen. Physiol. 121:529–540.1H, a cloned human T-type calcium channel. Am. J. Physiol. Heart Circ. Physiol. 278:H184–H193.
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M. L. Roberts-Crowley and A. R. Rittenhouse Arachidonic acid inhibition of L-type calcium (CaV1.3b) channels varies with accessory CaV{beta} subunits J. Gen. Physiol., April 1, 2009; 133(4): 387 - 403. [Abstract] [Full Text] [PDF]
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 K.-M. Fang, A.-S. Lee, M.-J. Su, C.-L. Lin, C.-L. Chien, and M.-L. Wu Free fatty acids act as endogenous ionophores, resulting in Na+ and Ca2+ influx and myocyte apoptosis Cardiovasc Res, June 1, 2008; 78(3): 533 - 545. [Abstract] [Full Text] [PDF]
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 S. J. Liu Inhibition of L-type Ca2+ channel current and negative inotropy induced by arachidonic acid in adult rat ventricular myocytes Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1594 - C1604. [Abstract] [Full Text] [PDF]
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 J. Chemin, J. Nargeot, and P. Lory Chemical Determinants Involved in Anandamide-induced Inhibition of T-type Calcium Channels J. Biol. Chem., January 26, 2007; 282(4): 2314 - 2323. [Abstract] [Full Text] [PDF]
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 J.-Y. Park, H.-W. Kang, H.-J. Moon, S.-U. Huh, S.-W. Jeong, N. M. Soldatov, and J.-H. Lee Activation of protein kinase C augments T-type Ca2+ channel activity without changing channel surface density J. Physiol., December 1, 2006; 577(2): 513 - 523. [Abstract] [Full Text] [PDF]
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 L. Liu, R. Zhao, Y. Bai, L. F. Stanish, J. E. Evans, M. J. Sanderson, J. V. Bonventre, and A. R. Rittenhouse M1 Muscarinic Receptors Inhibit L-type Ca2+ Current and M-Current by Divergent Signal Transduction Cascades. J. Neurosci., November 8, 2006; 26(45): 11588 - 11598. [Abstract] [Full Text] [PDF]
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 R. A. Gubitosi-Klug, D. J. Mancuso, and R. W. Gross The human Kv1.1 channel is palmitoylated, modulating voltage sensing: Identification of a palmitoylation consensus sequence PNAS, April 26, 2005; 102(17): 5964 - 5968. [Abstract] [Full Text] [PDF]
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