Inhibition of Kv4.3/KChIP2.2 Channels by Bupivacaine and Its Modulation by the Pore Mutation Kv4.3^V401I

Chinese hamster ovary cells were cultured in 50-ml flasks (NUNC, Roskilde, Denmark) at 37°C in Minimum Essential Medium Alpha medium (GIBCO; Invitrogen, Carlsbad, CA) with 10% fetal calf serum (Biochrom, Berlin, Germany), 100 U/ml penicillin, and 100 mg/ml streptomycin (GIBCO) in a humidified atmosphere (5% CO²). Cells were subcultured in 35-mm-diameter monodishes at least 1 day before transfection.

The mutant Kv4.3^V401Iwas created by site-directed mutagenesis. All constructs were cloned in the pcDNA3 expression vector. Chinese hamster ovary cells were transiently transfected with 0.1 μg Kv4.3^wt(GenBank No. NM 004980) or Kv4.3^V401Iplasmid DNA, 1.0 μg KChIP2.2 plasmid DNA (GenBank No. NM 173191), 0.3 μg EFGP cDNA, and 3 μl lipofectamine reagent (GIBCO) per dish according to the manufacturer’s protocol. Cells were cotransfected with an EGFP pcDNA3 construct to verify successful transfection. Only green fluorescing cells were used for patch clamp experiments. Patch clamp experiments were performed at room temperature 1–2 days after transfection.

Whole cell currents were measured with the patch clamp method28using an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany) and Pulse software version 8.11 (HEKA Elektronik). Patch electrodes with a pipette resistance between 2.0 and 4.0 MΩ were pulled from borosilicate glass capillary tubes (World Precision Instruments, Saratoga, FL) and filled with the following solution: 160 mm KCl, 0.5 mm MgCl², 10 mm HEPES, 2 mm Na-ATP (all from Sigma, Deissenhofen, Germany); pH 7.2 (adjusted with KOH). The external solution applied on the cells was of the following composition: 135 mm NaCl, 5 mm KCl, 2 mm CaCl², 2 mm MgCl², 5 mm HEPES, 10 mm sucrose, 0.1 mg/ml Phenol red (all from Sigma); pH 7.4 (adjusted with NaOH). The capacity of the cells was 29.8 ± 16.1 pF (n = 20). Series resistance was 2.5–5 MΩ and actively compensated for by at least 80%. A p/4 leak subtraction protocol was used in the study. Bupivacaine (Sigma), levobupivacaine (AstraZeneca, Södertalje, Sweden), and dextrobupivacaine (AstraZeneca) were dissolved in the extracellular solution. The drugs were superfused on the cells by a hydrostatically driven perfusion system.

The holding potential of the cell membrane was −80 mV, and Kv4.3 channels were activated by different protocols. For channel stimulation by the rectangle pulse protocol, the membrane was hyperpolarized for 200 ms to −100 mV and subsequently depolarized to +40 mV for 1,000 ms. Current activation was analyzed by depolarization of the membrane potential for 1,000 ms from −40 to +60 mV in 10-mV steps. Steady state inactivation was measured by depolarizing the membrane to +40 mV for 25 ms from prepulses of a duration of 4,000 ms increasing from −75 and −15 mV in steps of 5 mV. For measuring the recovery from inactivation, the initial depolarization of the membrane potential to +40 mV for 1,500 ms was followed by depolarizing the membrane potential to −80 mV for different duration of time increasing from 10 to 5,120 ms by doubling the time spent at −80 mV (Δt) after each individual depolarization. The repolarization was followed by a depolarizing step of 25 ms to + 40 mV. Experiments were performed at room temperature. The recorded signal was filtered at 2 kHz and stored on a personal computer for later analysis with a sampling rate of up to 20 kHz.

The current–time relation observed with the different protocols for potassium channel activation was used to determine the maximal outward current (I^max) as well as to quantify the charge (Q) crossing the membrane during the duration of the depolarization induced by the rectangle protocol. The charge crossing the membrane is equivalent to the time integrals of current traces and was determined using Pulse software version 8.11 (HEKA Elektronik). Inhibition was quantified by the ratio of time integrals of current traces in the presence of the drug to the mean of the time integrals before application of the drug and after washout of the drug (inhibition Q = 1 − Q^drug/[(Q^control+ Q^washout)/2]). The concentration–response curves were fitted with the Hill equation e/e^max= 1/[1 + (IC⁵⁰/c)^γ] using nonlinear regression (e = effect, e^max= maximal effect, c = anesthetic concentration, γ= Hill coefficient, and IC⁵⁰= concentration of half-maximal effect). Regression analysis was performed using Kaleidagraph (Synergy Software, Reading, PA). The whole cell conductance G^maxwas calculated using the following formula: G^max= I^max/(V^m− E^K), where I^maxis the maximum current during each test potential, V^mis the membrane potential, and E^Kis the Nernst potential for potassium (−87.54 mV under our experimental conditions). The means of the whole cell conductance were mathematically described by a Boltzmann function G = G^max/[1 + exp((V^0.5− V^m)/k)], where G^maxis the maximal whole cell conductance, V^0.5is the voltage of half-maximal activation, V^mis the membrane potential, and k is the slope factor, using Kaleidagraph software. Time-dependent inactivation was mathematically described using a biexponential function y = C + A¹exp(−t/τ¹) + A²exp(−t/τ²), where τ¹and τ²are the system time constants, A¹and A²are the amplitudes of each component of the exponential, and C is the baseline value. Recovery from inactivation was determined by relating the maximal size of the outward current during the test pulse to the size of maximal outward current during the prepulse. The time-dependent increase of this ratio was mathematically described with a monoexponential or biexponential function as described above. The time course of the development of block was analyzed using the following equation: y = (I^control− I^drug)/I^control, where I^controlis the current at any given time under control conditions and I^drugis the current at the same time under drug conditions. Data points are presented as mean ± SD unless stated otherwise. Statistical significance was tested using analysis of variance and the Tukey-Kramer multiple comparisons test (Graph Pad; Prism, San Diego, CA) or two-sided paired and unpaired Student t  tests as appropriate (Excel; Microsoft, Redmond, WA). A value of P < 0.05 was regarded as significant. The number n is the number of experiments.

Kv4.3^wt/KChIP2.2 currents activated with a midpoint of 0 ± 4 mV (n = 10) and were fully activated at a potential of +40 mV. Therefore, a rectangle protocol with a depolarization to +40 mV was chosen to test the influence of bupivacaine and levobupivacaine on the channel complexes. The rectangle protocol evoked a rapidly activating and inactivating A-type current (fig. 1A). Bupivacaine (30 μm) had two effects on the Kv4.3^wt/KChIP2.2 current. It reduced the current, and it accelerated macroscopic current decline in a such way that a crossing phenomenon occurred upon overlay of control and drug traces. Both effects were reversible upon washout (fig. 1A). The current decline of Kv4.3^wt/KChIP2.2 currents was described using two time constants. Bupivacaine, 30 μm, reduced the fast time constant τ¹from 35 ± 14 ms (n = 9) to 10 ± 3 ms (n = 5; P < 0.01), and it increased the slow time constant τ²from 96 ± 44 ms (n = 9) to 147 ± 37 ms (n = 5; P < 0.05). The ratio of the amplitudes of the time constants, amplitude 1 and amplitude 2, was changed by bupivacaine from 62 ± 14% for amplitude 1 and 38 ± 14% for amplitude 2 under control and washout conditions to 80 ± 5% for amplitude 1 and 20 ± 5% for amplitude 2 when 30 μm bupivacaine was applied (n = 5; P < 0.05). This resulted in the crossing phenomenon. Consequently, bupivacaine action on the channel complex was inhibitory before the crossing but stimulatory after the crossing of currents.

Figure 1Bshows the effect of different concentrations of bupivacaine on the Kv4.3^wt/KChIP2.2 channel complex. The reduction of current and the change of current shape were concentration dependent. The inhibition of Kv4.3^wt/KChIP2.2 channels was quantified as reduction of the charge transfer through Kv4.3^wt/KChIP2.2 channels during the entire time of the depolarization (Q) as well as reduction of the charge transfer from the beginning of the depolarization until the crossing of currents (Q^cross). The concentration–response data were mathematically described with Hill functions (fig. 1C). The IC⁵⁰values for inhibition of Q, and Q^crossby bupivacaine were 55 ± 8 μm (n = 20) and 30 ± 7 μm (n = 24), respectively (table 1). The acceleration of macroscopic current decline was concentration-dependent as well, and the time until the crossing occurred increased with concentration (fig. 1B). The Hill function describing the concentration–response data for reduction of τ¹yielded an IC⁵⁰value of 17 ± 6 μm (n = 26) (fig. 1Dand table 1). Although τ²was increased by 30 μm bupivacaine, it was impossible to be analyzed under higher drug concentrations (300 μm and 1 mm) because of the small size of the residual current. Levobupivacaine had similar effects on Kv4.3^wt/KChIP2.2. The IC⁵⁰values for inhibition of Q, Q^cross, and reduction of τ¹by levobupivacaine were 50 ± 5 μm (n = 26), 26 ± 2 μm (n = 28), and 17 ± 1 μm (n = 29), respectively (table 1). There was no significant difference in any parameter of Kv4.3^wt/KChIP2.2 inhibition by racemic bupivacaine and by levobupivacaine (P > 0.05).

To further analyze the possible stereospecific effect of bupivacaine, paired tests were performed with the pure enantiomers S  (−)-levobupivacaine and R  (+)-dextrobupivacaine at a concentration of 30 μm. This concentration was chosen because it was near the IC⁵⁰value for the inhibition of Q and hence at the steepest part of the concentration–response curve. A gentle difference in potency between the enantiomers should best be detectable at this part of the concentration–response curve. Levobupivacaine and dextrobupivacaine inhibited Q at 30 μm with nearly identical potencies (levobupivacaine: 51.4 ± 1%, dextrobupivacaine: 50.5 ± 1%; n = 11; P > 0.05). Therefore, only racemic bupivacaine was used in the following experiments.

To establish the effect of bupivacaine on Kv4.3^wt/KChIP2.2 channel gating, the effects of 100 μm bupivacaine on voltage-dependent activation, steady state inactivation, and recovery from inactivation were investigated (figs. 2 and 3). Bupivacaine affected voltage-dependent activation as well as recovery from inactivation without changing steady state inactivation. Figure 2shows original current traces elicited by the activation protocol (fig. 2A), the inactivation protocol (fig. 2B), and the recovery protocol (fig. 2C). Application of 100 μm bupivacaine reduced the current during every protocol. This effect was always reversible upon washout. Bupivacaine (100 μm) modulated the voltage dependence of activation by shifting the midpoint of current activation in the depolarizing direction by 10 ± 4 mV (n = 5; P < 0.05). The slope of the activation curve was increased by 2 ± 1 mV (n = 5; P < 0.05; fig. 3Aand table 2). The voltage dependence of inactivation was not altered by 100 μm bupivacaine (fig. 3B). However, the local anesthetic slowed the recovery from inactivation (fig. 3C). Under control and washout conditions, the time dependence of the recovery from inactivation was adequately fitted with one time constant (τ^rec, 57 ± 7 ms; n = 6). Application of 100 μm bupivacaine altered Kv4.3/KChIP2.2 gating in such a way that two time constants were necessary to adequately describe the time dependence of recovery (τ^rec1= 42 ± 6 ms, τ^rec2= 2,339 ± 728 ms; n = 6). Both time constants differed significantly from the time constant under control and washout conditions (P < 0.05; table 2)

Voltage dependence of Kv4.3^wt/KChIP2.2 block was analyzed as the inhibition of Q. Inhibition of Q by 100 μm bupivacaine increased between membrane potentials of −10 and +40 mV in a voltage-dependent manner (fig. 3D; P < 0.05, tested by analysis of variance). At −10 mV, inhibition of Q was 67 ± 3% whereas at +40 mV, Q was inhibited by 74 ± 2% (n = 4). The fast time constant of current decline (τ¹) was inversely related to voltage under control as well as under bupivacaine (100 μm) conditions (fig. 3E; P < 0.05, tested by analysis of variance). Under control conditions, τ¹was 48 ± 10 ms at −10 mV and 33 ± 3 ms at +40 mV (n = 4). When bupivacaine was applied, τ¹was 18 ± 8 ms at −10 mV and decreased to 5 ± 1 ms at +40 mV (n = 4; fig. 3E). Because it was not always possible to describe the inactivation process with two time constants at −10 mV, the voltage dependence of the amplitudes was analyzed between membrane potentials of 0 mV and +40 mV. The distribution of the amplitudes of the fast and the slow time constant of current decline was voltage-dependent under control and washout conditions (fig. 3F), with values of 86 ± 9% for amplitude 1 and 14 ± 9% for amplitude 2 at 0 mV and 65 ± 5% for amplitude 1 and 35 ± 5% for amplitude 2 at +40 mV. Application of 100 μm bupivacaine reversed voltage dependence of distribution of the amplitudes, with an amplitude 1 of 79 ± 7% at 0 mV and 87 ± 7 at +40 mV (n = 4; P < 0.05).

To gain further insight into the mechanism of Kv4.3 inhibition by bupivacaine, we in addition analyzed the pore mutant Kv4.3^V401I. The electrophysiologic properties of this pore mutant were characterized first. Figures 4A and Bshow normalized currents through Kv4.3 wild-type and mutant channels expressed with and without KChIP2.2. For Kv4.3^wt, a crossing of current traces with and without KChIP is observed (fig. 4A). In contrast, no such crossing is found for Kv4.3^V401(fig. 4B). The mutant channel Kv4.3^V401Iexhibited a slower macroscopic current decline than Kv4.3^wt(figs. 4A and B). τ¹was 20 ± 2 ms (n = 10) for Kv4.3^wtand 33 ± 6 ms (n = 7) for Kv4.3^V401I(P < 0.01; fig. 4Cand table 3). The slow time constant τ²was also slowed by the mutation (130 ± 25 ms for Kv4.3^wt, n = 10, vs.  293 ± 48 ms for Kv4.3^V401I, n = 7; P < 0.01; table 3). KChIP2.2 reduced τ²but had no effect on the fast time constant of Kv4.3^V401Icurrent decline (figs. 4B and Cand table 3). KChIP2.2 increased the current density of both Kv4.3^wtas well as Kv4.3^V401Ichannels (fig. 4D). Current densities did not significantly differ between Kv4.3^wtand Kv4.3^V401I. This was also the case for Kv4.3^wtand Kv4.3^V401Iwhen expressed in combination with KChIP2.2 (fig. 4D). The voltage dependence of activation and inactivation of Kv4.3^V401Iwere similar to Kv4.3^wt. The voltage dependence of inactivation was shifted to more negative potentials by coexpression with KChIP2.2 (fig. 4E; activation: V^0.5= 7.7 ± 6.0 mV for Kv4.3^V401I, n = 9, and V^0.5= 6.0 ± 7.4 mV for Kv4.3^V401I/KChIP2.2, n = 7, P > 0.05; inactivation: V^0.5=−46.6 ± 2.6 mV for Kv4.3^V401I, n = 7, and V^0.5=−52.6 ± 2.0 mV for Kv4.3^V401I/KChIP2.2, n = 6; P < 0.05). The mutation significantly slowed the recovery from inactivation of Kv4.3 channels (compare figs. 3C and 4F). Coexpression of KChIP2.2 did not significantly change the time constants of recovery from inactivation (fig. 4F; τ^rec= 250 ± 19 ms for Kv4.3^V401I, n = 5, and τ^rec= 240 ± 48 ms for Kv4.3^V401I/KChIP2.2, n = 5; P > 0.05).

To investigate whether the accessory β subunit KChIP2.2 modulates bupivacaine sensitivity of Kv4.3 channels, the inhibition of Kv4.3^wt/KChIP2.2 channel complexes by bupivacaine (30 μm) was compared with the inhibition of Kv4.3^wtchannels expressed without the β subunit as well as with inhibition of the pore mutant Kv4.3^V401Iexpressed with and without KChIP2.2 (figs. 5A–C). Bupivacaine (30 μm) inhibited Kv4.3^wt, Kv4.3^V401I, and Kv4.3^V401I/KChIP2.2 channels (figs. 5A–C). However, in contrast to Kv4.3^wt/KChIP2.2, bupivacaine did not induce a crossing phenomenon in Kv4.3^wt, Kv4.3^V401I,or Kv4.3^V401I/KChIP2.2 channels (compare figs. 5A–Cwith fig. 1A). As a consequence, inhibition (Q) of Kv4.3^wt/KChIP2.2 was significantly less than inhibition (Q) of Kv4.3^wt(36 ± 6% for Kv4.3^wt/KChIP2.2, n = 5, vs.  54 ± 5% for Kv4.3^wt, n = 10; P < 0.01; fig. 5D). The reduction of bupivacaine sensitivity of Kv4.3^wtby KChIP2.2 was not observed with the pore mutant Kv4.3^V401I. Inhibition (Q) of Kv4.3^V401Iamounted to 53 ± 9% (n = 6) in the absence of KChIP2.2, and it was 50 ± 7% (n = 7) in the presence of KChIP2.2 (P > 0.05; fig. 5D). Inhibition of Kv4.3^V401Iand Kv4.3^wtchannels did not significantly differ (P > 0.05; fig. 5D). KChIP2.2 slowed the development of block by bupivacaine of Kv4.3^wtchannels but not of Kv4.3^V401Ichannels. The time constant for the development of Kv4.3^wtblock was 3 ± 1 ms (n = 6), 6 ± 1 ms for Kv4.3^wt/KChIP2.2 (n = 5), 4 ± 1 ms for Kv4.3^V401I(n = 6), and 4 ± 1 ms for Kv4.3^V401I/KChIP2.2 (n = 8). Block of both Kv4.3^V401Iand Kv4.3^V401I/KChIP2.2 channels developed significantly slower than block of Kv4.3^wt/KChIP2.2 channels (P < 0.01).

This study established the effects of the local anesthetic bupivacaine on Kv4.3^wt/KChIP2.2 and Kv4.3^V401I/KChIP2.2 channel complexes expressed in Chinese hamster ovary cells. The biophysical properties of the wild-type ion channel complex were in accord with biophysical properties of Kv4/KChIP2 channel complexes reported previously.11,13The local anesthetics bupivacaine and levobupivacaine reduced Kv4.3^wt/KChIP2.2 currents in a concentration-dependent and reversible manner. Both drugs exhibited the same effects on Kv4.3^wt/KChIP2.2 and did not differ with regard to inhibitory potency. Bupivacaine caused a depolarizing shift of voltage-dependent current activation. The local anesthetic decreased the time constants of macroscopic current decline, slowed recovery from channel inactivation, and induced a crossing phenomenon of currents.

Acceleration of macroscopic current decline is a characteristic feature of open channel block.29,30This mechanism has already been described previously for the inhibition of several Kv α subunits, including Kv4.3 by bupivacaine.17,19,20,31,32Inhibition of Kv1.5 channels by bupivacaine is mediated by threonine 477 (current number according to GenBank No. NM 002234: T479) within the tetraethylammonium binding domain in the pore region of Kv1.5 channels and also by threonine 505, lysine 508, and valine 512 in the S6 segment of Kv1.5 channels17(current numbers according to GenBank No. NM 002234: T507, K510, V514). These amino acids are also important for the stereoselective inhibition of Kv1.5 channels by bupivacaine. Mutation of T505 to valine effectively reduces stereoselectivity.17The corresponding position in Kv4.3 channels is valine 394. Therefore, the results of our study are in accord with the previous mutagenesis studies on Kv1.5,17as well as with results obtained with native I^to.26However, in contrast to bupivacaine, ropivacaine interacted with Kv4.3/KChIP2.2 channels in a stereoselective manner.24The lipophilicity of these local anesthetics may therefore be inversely related to their ability of stereospecific molecular interaction with Kv4.3/KChIP2.2 channels. Further study is needed to finally resolve this issue.

The crossing of currents under drug condition with the currents under drug free condition may be explained by a reversal of the effects of KChIP2.2 on Kv4.3^wtchannel gating.24A possible interaction of bupivacaine with KChIP2.2 may also explain why the block of Kv4.3^wt/KChIP2.2 developed significantly slower than block of Kv4.3^wtchannels. Because KChIP2.2 changes the inhibition of Kv4.3^wtchannels by the open pore blocker bupivacaine, it could be further hypothesized that KChIP2.2 also binds to the pore region. However, this seems less likely for several reasons.

It is well established that the N-terminus is crucial for KChIP interaction with Kv4 channels.9,11By impairing the movement of the N-termini to the internal mouth of the pore, KChIPs reduce N-type inactivation from the open state33and increase closed state inactivation, resulting from conformational changes at the internal vestibule of the pore (V-type inactivation).33,34This effect of KChIP can be antagonized by intracellular application of an N-terminal peptide.35As these changes in channel gating occur without a direct interaction of KChIP with the ion channel pore, sterical interaction of the pore blocker bupivacaine and KChIP2.2 is unlikely to occur. This view is supported by the results obtained with the Kv4.3 pore mutant V401I. The mutation V401I slows macroscopic current decline of Kv4.3/KChIP2.2 channel complexes as well as recovery from inactivation. As the current density of both Kv4.3^wtand Kv4.3^V401Ichannels is increased by coexpression with KChIP2.2, binding of KChIP2.2 to Kv4.3^V401Ichannels does not seem impaired. In addition, the voltage dependence of activation and inactivation of Kv4.3^V401Iis very similar to that of Kv4.3^wt. Hence, the gating properties of the channel and of the channel complex with KChIP2.2 are only marginally influenced by this mutation, making it particularly suitable for pharmacologic studies. In contrast to Kv4.3^wtchannels, inhibition of Kv4.3^V401Ichannels by bupivacaine is not reduced by coexpression with KChIP2.2, and bupivacaine does not induce a crossing of inhibited currents with control currents. Furthermore, the onset of block of Kv4.3^V401Iis not altered by coexpression with KChIP2.2. The lack of a crossing phenomenon may be explained by different inactivation time constants of Kv4.3^wt/KChIP2.2 and Kv4.3^V401I/KChIP2.2. τ¹of current decline is significantly decreased by the local anesthetic in all channels investigated, whereas τ²is significantly increased by bupivacaine only in Kv4.3^wt/KChIP2.2 complexes. When comparing the absolute values of the time constants of current decline and their amplitudes (table 3), it becomes obvious that despite different inactivation time constants under control conditions, both τ¹and τ²of Kv4.3^wt/KChIP2.2 and Kv4.3^V401I/KChIP2.2 current decline behave in exactly the same way under the influence of bupivacaine. The reduction of Kv4.3 inhibition caused by coexpression with KChIP2.2, therefore, is a consequence of the relatively small τ²value of Kv4.3^wt/KChIP2.2 under control conditions. Because KChIP2.2 binds to both Kv4.3^wtas well as Kv4.3^V401I, the interaction of bupivacaine with the ion channel pore is unlikely to be altered by direct interaction of the local anesthetic with KChIP2.2.

The results of this study add further evidence to the model suggested previously to describe the interference of effects of amino-amide local anesthetics and KChIP subunits while acting on Kv4.3 channels.24Kv4.3 channels inactivate from a preopen closed state by a mechanism involving conformational changes at the internal vestibule of the pore33,34as well as from the open state by a mechanism involving the N-termini.33,35The interaction of KChIP with the N-termini impairs inactivation from the open state by preventing the channel’s N-termini to interact with the internal mouth of the pore.33Consequently, inactivation from the preopen closed state predominates in complexes formed by Kv4 and KChIP subunits.33By interacting with the channel pore, bupivacaine introduces a blocked open state of Kv4.3/KChIP2.2 channels reminiscent of N-type inactivation.29,30Bupivacaine thus reverses KChIP2.2 effects on Kv4.3 channels by mimicking the interaction of the N-termini of the α subunit with its ion channel pore.

In summary, the amino-amide local anesthetic bupivacaine inhibited complexes formed by Kv4.3/KChIP2.2 in a concentration-dependent and reversible manner. The results of our study are consistent with the idea that bupivacaine blocks Kv4.3/KChIP2.2 channels from the open state. By influencing channel gating, KChIP2.2 indirectly altered the response of Kv4.3 channels to bupivacaine.

The authors thank Andrea Zaisser (Technician, Institute of Neuronal Signal Transduction, Center for Molecular Neurobiology, University of Hamburg, Hamburg, Germany) for cell culture; Dirk Isbrandt, M.D. (Research Group Leader), and Kathrin Sauter (Technician, both from the Institute of Neuronal Signal Transduction) for providing the Kv4.3 mutant V401I; Dirk Isbrandt, M.D., and Robert Bähring, Ph.D. (Privatdozent, Institute of Neuronal Signal Transduction), for critically reading the manuscript; Robert Bähring, Ph.D., for help with the gating model; and Olaf Pongs, Ph.D. (Director, Institute of Neuronal Signal Transduction), for continuous support.