The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated.
Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels.
Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 +/- 8 and 50 +/- 5 microm, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2.
These results indicate that bupivacaine blocks Kv4.3/KChIP2.2 channels from the open state. They furthermore give structural evidence that amino-amide local anesthetics interfere with the effects of KChIP2.2 on Kv4.3 by an indirect mechanism.
THE transient outward current Itois an important repolarizing K current in the human heart.1,2The reduction of Itocontributes to prolongation of the cardiac action potential and ventricular arrhythmia occurring in ventricular failure3,4and in myocardial infarction.5Although the exact molecular composition of human Itoin vivo remains unclear,6it is widely accepted that Kv4.3 constitutes the principal α subunit in the human heart.1,6–8Kv4.3 α subunits coassemble with accessory subunits of the KChIP2 family (Kv channel interacting protein).9,10KChIP2 alters the level of surface expression as well as the gating properties of Kv4 channels.11The level of KChIP2 gene expression decreases from the epicardium to the endocardium and correlates with the transmural gradient of Itoin canine,7,8murine,12and human myocardium.7,8Knockout of the KChIP2 gene in mice leads to a complete absence of Ito, a prolongation of action potential duration, and an increased susceptibility to development of polymorphic ventricular arrhythmia of the torsades de pointes type.12The precise mechanism of the interaction of KChIP with Kv4 channels has not been elucidated. However, it is clear that the N-terminal part of the Kv4.3 α subunit is crucial in binding and interacting with KChIP2.11The amino acids valine 399 and valine 401 in the inner pore region of Kv4.3 channels are also considered important for the modulatory effects of KChIP2.13
Inhibition of K channels by bupivacaine has been regarded as the molecular mechanism responsible for QTc interval prolongation of the electrocardiogram observed during toxic plasma concentrations of this amino-amide local anesthetic.14–16The pharmacologic effects of bupivacaine on cloned cardiac repolarizing K channel α subunits have been studied.17–24Also, the pharmacologic action of bupivacaine on some cardiac ion channel complexes formed from α and β subunits has been investigated.18,22,24The results of these studies demonstrate that amino-amide local anesthetics are not selective Na channel blockers but also block potassium channels in the same concentration range. In addition, these studies propose that amino-amide local anesthetics act as open channel blockers of Kv1.5 and Kv4.3 channels17,20,24and that local anesthetics interfere with the effects of KChIP on Kv4.3 channels by an unknown mechanism.24
Itois suggested to constitute a potential molecular target involved in the cardiotoxic action of bupivacaine.25,26However, the effects of bupivacaine on complexes formed by human Kv4 and KChIP subunits have not yet been studied. Furthermore, it is unknown whether local anesthetics directly or indirectly interfere with the action of KChIP subunits by binding to the ion channel pore of Kv4.3 channels. Kv4.3 and KChIP2.2 constitute the most abundant isoforms of Kv4 and KChIP subunits present in human heart.11,27Therefore, this study was designed to investigate in detail bupivacaine effects on complexes formed by Kv4.3 and KChIP2.2 subunits cloned from human heart. Investigating the effects of bupivacaine on a pore mutation of Kv4.3 channels predicted to alter interaction with KChIP2.213could also demonstrate whether bupivacaine and KChIP interfere by a direct or indirect mechanism.
Materials and Methods
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.
Molecular Biology and Transfection of Cells
The mutant Kv4.3V401Iwas 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 Kv4.3wt(GenBank No. NM 004980) or Kv4.3V401Iplasmid 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.
Electrophysiology of Potassium Channels
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 (Imax) 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 − Qdrug/[(Qcontrol+ Qwashout)/2]). The concentration–response curves were fitted with the Hill equation e/emax= 1/[1 + (IC50/c)γ] using nonlinear regression (e = effect, emax= 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 Gmaxwas calculated using the following formula: Gmax= Imax/(Vm− EK), where Imaxis the maximum current during each test potential, Vmis the membrane potential, and EKis 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 = Gmax/[1 + exp((V0.5− Vm)/k)], where Gmaxis the maximal whole cell conductance, V0.5is the voltage of half-maximal activation, Vmis 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 = (Icontrol− Idrug)/Icontrol, where Icontrolis the current at any given time under control conditions and Idrugis 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.
Kv4.3wt/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 Kv4.3wt/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 Kv4.3wt/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 Kv4.3wt/KChIP2.2 channel complex. The reduction of current and the change of current shape were concentration dependent. The inhibition of Kv4.3wt/KChIP2.2 channels was quantified as reduction of the charge transfer through Kv4.3wt/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 (Qcross). The concentration–response data were mathematically described with Hill functions (fig. 1C). The IC50values for inhibition of Q, and Qcrossby 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 Kv4.3wt/KChIP2.2. The IC50values for inhibition of Q, Qcross, 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 Kv4.3wt/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 Kv4.3wt/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 (τrec, 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 (τrec1= 42 ± 6 ms, τrec2= 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 Kv4.3wt/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 Kv4.3V401I. 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 Kv4.3wt, a crossing of current traces with and without KChIP is observed (fig. 4A). In contrast, no such crossing is found for Kv4.3V401(fig. 4B). The mutant channel Kv4.3V401Iexhibited a slower macroscopic current decline than Kv4.3wt(figs. 4A and B). τ1was 20 ± 2 ms (n = 10) for Kv4.3wtand 33 ± 6 ms (n = 7) for Kv4.3V401I(P < 0.01; fig. 4Cand table 3). The slow time constant τ2was also slowed by the mutation (130 ± 25 ms for Kv4.3wt, n = 10, vs. 293 ± 48 ms for Kv4.3V401I, n = 7; P < 0.01; table 3). KChIP2.2 reduced τ2but had no effect on the fast time constant of Kv4.3V401Icurrent decline (figs. 4B and Cand table 3). KChIP2.2 increased the current density of both Kv4.3wtas well as Kv4.3V401Ichannels (fig. 4D). Current densities did not significantly differ between Kv4.3wtand Kv4.3V401I. This was also the case for Kv4.3wtand Kv4.3V401Iwhen expressed in combination with KChIP2.2 (fig. 4D). The voltage dependence of activation and inactivation of Kv4.3V401Iwere similar to Kv4.3wt. The voltage dependence of inactivation was shifted to more negative potentials by coexpression with KChIP2.2 (fig. 4E; activation: V0.5= 7.7 ± 6.0 mV for Kv4.3V401I, n = 9, and V0.5= 6.0 ± 7.4 mV for Kv4.3V401I/KChIP2.2, n = 7, P > 0.05; inactivation: V0.5=−46.6 ± 2.6 mV for Kv4.3V401I, n = 7, and V0.5=−52.6 ± 2.0 mV for Kv4.3V401I/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; τrec= 250 ± 19 ms for Kv4.3V401I, n = 5, and τrec= 240 ± 48 ms for Kv4.3V401I/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 Kv4.3wt/KChIP2.2 channel complexes by bupivacaine (30 μm) was compared with the inhibition of Kv4.3wtchannels expressed without the β subunit as well as with inhibition of the pore mutant Kv4.3V401Iexpressed with and without KChIP2.2 (figs. 5A–C). Bupivacaine (30 μm) inhibited Kv4.3wt, Kv4.3V401I, and Kv4.3V401I/KChIP2.2 channels (figs. 5A–C). However, in contrast to Kv4.3wt/KChIP2.2, bupivacaine did not induce a crossing phenomenon in Kv4.3wt, Kv4.3V401I,or Kv4.3V401I/KChIP2.2 channels (compare figs. 5A–Cwith fig. 1A). As a consequence, inhibition (Q) of Kv4.3wt/KChIP2.2 was significantly less than inhibition (Q) of Kv4.3wt(36 ± 6% for Kv4.3wt/KChIP2.2, n = 5, vs. 54 ± 5% for Kv4.3wt, n = 10; P < 0.01; fig. 5D). The reduction of bupivacaine sensitivity of Kv4.3wtby KChIP2.2 was not observed with the pore mutant Kv4.3V401I. Inhibition (Q) of Kv4.3V401Iamounted 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 Kv4.3V401Iand Kv4.3wtchannels did not significantly differ (P > 0.05; fig. 5D). KChIP2.2 slowed the development of block by bupivacaine of Kv4.3wtchannels but not of Kv4.3V401Ichannels. The time constant for the development of Kv4.3wtblock was 3 ± 1 ms (n = 6), 6 ± 1 ms for Kv4.3wt/KChIP2.2 (n = 5), 4 ± 1 ms for Kv4.3V401I(n = 6), and 4 ± 1 ms for Kv4.3V401I/KChIP2.2 (n = 8). Block of both Kv4.3V401Iand Kv4.3V401I/KChIP2.2 channels developed significantly slower than block of Kv4.3wt/KChIP2.2 channels (P < 0.01).
This study established the effects of the local anesthetic bupivacaine on Kv4.3wt/KChIP2.2 and Kv4.3V401I/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 Kv4.3wt/KChIP2.2 currents in a concentration-dependent and reversible manner. Both drugs exhibited the same effects on Kv4.3wt/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 Ito.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 Kv4.3wtchannel gating.24A possible interaction of bupivacaine with KChIP2.2 may also explain why the block of Kv4.3wt/KChIP2.2 developed significantly slower than block of Kv4.3wtchannels. Because KChIP2.2 changes the inhibition of Kv4.3wtchannels 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 Kv4.3wtand Kv4.3V401Ichannels is increased by coexpression with KChIP2.2, binding of KChIP2.2 to Kv4.3V401Ichannels does not seem impaired. In addition, the voltage dependence of activation and inactivation of Kv4.3V401Iis very similar to that of Kv4.3wt. 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 Kv4.3wtchannels, inhibition of Kv4.3V401Ichannels 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 Kv4.3V401Iis not altered by coexpression with KChIP2.2. The lack of a crossing phenomenon may be explained by different inactivation time constants of Kv4.3wt/KChIP2.2 and Kv4.3V401I/KChIP2.2. τ1of current decline is significantly decreased by the local anesthetic in all channels investigated, whereas τ2is significantly increased by bupivacaine only in Kv4.3wt/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 Kv4.3wt/KChIP2.2 and Kv4.3V401I/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 Kv4.3wt/KChIP2.2 under control conditions. Because KChIP2.2 binds to both Kv4.3wtas well as Kv4.3V401I, 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.