INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

ACTION POTENTIAL WAVEFORMS differ between atrial and ventricular myocytes as well as between subepicardial and subendocardial myocytes, as shown for many species including dog, rat, and human (3, 14). This heterogeneity can be traced back to differences in outward current (2, 21, 25, 42). In particular, the transient outward current (I_to) is more prominent in subepicardial than in subendocardial myocytes (rat ventricle; see Ref. 14). In rat ventricular myocytes two components of outward current are distinguished kinetically and pharmacologically (4, 14, 40), the rapidly activating and inactivating I_to, which is sensitive to blockade by 4-aminopyridine (4-AP), and the rapidly activating but slowly inactivating delayed-rectifier-like current (I_K), which can be blocked by tetraethylammonium (TEA). Furthermore, I_to and I_K differ with respect to the potential dependence of availability of the underlying channels (4). In some cardiac preparations, e.g., Purkinje fiber, dog ventricle, and human atrium, I_to can be subdivided into a cytosolic Ca²⁺-insensitive, 4-AP-sensitive I_to1 and a cytosolic Ca²⁺-sensitive, 4-AP-insensitive I_to2 (6). In other preparations such as rat ventricle, only the cytosolic Ca²⁺-insensitive, 4-AP-sensitive I_to1 has been described (23). In the present paper, only I_to1 is considered, and for reasons of simplicity it is referred to as I_to.

With molecular biological approaches, a multitude of depolarization-activated K⁺-channel genes of the Kv1, Kv2, and Kv4 families and rat erg and KvLQT1 have been identified in adult and embryonic rat ventricle, respectively (7, 16, 17). Among those genes, Kv4.2 and Kv4.3 encode for K⁺-channel proteins with I_to-like properties (19, 39, 45), whereas three other gene products (Kv1.2, Kv1.5, Kv2.1) are channel proteins with I_K-like properties (9, 20, 22, 28, 36, 44). On the basis of these reports, more than two components of K⁺ outward current of rat ventricular myocytes are expected to be distinguishable, provided that the gene products differ in electrophysiological and/or pharmacological properties.

Here we report that it is in fact possible to differentiate at least four components of outward current by making use of steady-state inactivation kinetics. The sensitivity of these components to block by TEA, 4-AP, dendrotoxin (DTX), heteropodatoxin (HpTx3), and hanatoxin and other tools yielded pharmacological profiles that were compared with those reported for cloned channels. Because the amplitude of total outward current declines from subepicardial to subendocardial cells within the ventricular wall, we have also characterized the contribution of each current component to this differential current distribution. Preliminary results have been published in abstract form (22a and 22b).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cell isolation. All studies complied with the German home office regulations governing the care and use of laboratory animals. Male Wistar rats (body wt 200-250 g) were killed by cervical dislocation. As described previously (41), the hearts were perfused on a Langendorff apparatus at 37°C for 5-7 min with nominally Ca²⁺-free saline solution (composition in mM: 100 NaCl, 10 KCl, 5.0 MgSO₄, 1.2 KH₂PO₄, 20 glucose, 50 taurine, and 5.0 MOPS, adjusted to pH 7.0 with NaOH). The rat hearts were then perfused for 15 min with collagenase-containing solution (collagenase type I, 0.5 g/l, Sigma C-0130, Munich, Germany) supplemented with CaCl₂ (200 µM) and albumin (1 g/l). After enzyme perfusion, the hearts were chopped into small pieces and dissociation was continued by gentle stirring of the tissue pieces in fresh enzyme solution for 5-15 min. On some occasions tissue batches were dissected from the apex and the base of the heart, and their dissociation was continued separately to obtain subepicardial and subendocardial myocytes, respectively (14). Single ventricular myocytes were collected in a low-Ca²⁺ solution, the Ca²⁺ concentration of which was slowly increased (0.2 mM steps in intervals of 10 min) until a final concentration of 0.6 mM was reached. The cells were stored at room temperature and used within 12 h.

Whole cell voltage-clamp technique. Myocytes were transferred to a small Perspex chamber (volume 0.5 ml) placed on the stage of an inverted microscope (Olympus IMT-2 or Zeiss Axiovert-10). The chamber was continuously perfused at a constant rate (1.2 ml/min). Only rod-shaped myocytes with clear striations were used. For action potential and membrane current recordings, the single-electrode voltage-clamp technique was applied. Heat-polished pipettes made from borosilicate filament glass (OD 1.5 mm, Hilgenberg, Malsfeld, Germany) were used to form gigaohm seals with gentle suction; on average, the seal resistance was 2.8 G (range 1-12 G). The patched membrane was then disrupted by a pulse of suction to establish continuity of the interior of the electrode with the cytosol. Voltage or current clamp was achieved using a List L/M-EPC-7 or an Axopatch 200 amplifier. For stimulus protocol design and data acquisition, the Axolab TL-125 interface and pCLAMP 5.5 software (Axon Instruments, Foster City, CA) were used.

Measurement of action potentials. Action potentials in rat myocytes were measured in the current-clamp mode after injection of current (duration 3-5 ms, amplitude 0.8-1.0 nA, stimulation rate 0.1 Hz, room temperature). For these experiments, pipettes were pulled with tip resistances of 3.0-5.0 M when filled with a solution containing (in mM) 140 KCl, 4.0 MgCl₂, 5.0 CaCl₂, 10.0 EGTA, 10.0 HEPES, and 4.0 Na₂ATP, adjusted to pH 7.3 with KOH. Thus the free Ca²⁺ concentration was buffered to 50 nM (free Mg²⁺ concentration 300 µM) as calculated by the computer program EQCAL (Biosoft, Cambridge, UK). The bath solution was composed of (in mM) 150 NaCl, 5.4 KCl, 2.0 MgCl₂, 10 glucose, 10 HEPES, and 1.2 CaCl₂, adjusted to pH 7.4 with NaOH. Action potentials were analyzed for RMP and action potential amplitude. Action potential duration (APD) was measured at 20, 50, and 90% of repolarization (APD₂₀, APD₅₀, APD₉₀).

Measurement of outward K⁺ current. To measure outward current in ventricular myocytes of rat heart, the bath was perfused with a solution similar to that for action potential recording, except that 0.6 mM CaCl₂ and 0.1 mM CdCl₂ were used to block Ca²⁺ channels. Electrodes had tip resistances of 1.5-2.5 M when filled with the same solution as used for action potential recordings. Current-voltage relations (range 40 to +60 mV) were measured with 300-ms clamp steps in 10-mV increments after Na⁺-current inactivation by a 40-ms clamp step to 40 mV from the holding potential of 80 mV. For steady-state inactivation, 2,000-ms conditioning clamp steps (range 140 to +20 mV; 10-mV increments) were followed by a test clamp step to +60 mV (duration 300 ms); a step of 5 ms at 40 mV was interposed between conditioning and test clamp steps to keep step amplitude and capacitive current constant.

Chemicals. Samples of HpTx3 and hanatoxin were kindly provided by NPS Pharmaceuticals (Salt Lake City, UT) and Dr. Kenton Swartz (National Institutes of Health, Bethesda, MD), respectively. Clofilium tosylate was a gift of Eli Lilly (Indianapolis, IN), and quinidine hemisulfate was from Merck (Darmstadt, Germany). All drugs were dissolved in H₂O; aliquots of concentrated stock solutions were stored at 20 C until use. Enzymes used for cell isolation (collagenase type I) and BSA were obtained from Sigma Chemicals. All other chemicals were purchased from commercial suppliers and were of laboratory grade.

Data analysis. Steady-state inactivation curves were obtained by plotting normalized current (I/I_max) at the test potential as a function of the conditioning potential (V_m) and fitting a Boltzmann function to the data points where V_0.5 and k are the potentials of half-maximal inactivation and the slope factor, respectively. However, a single Boltzmann function did not adequately describe the data, whereas in almost all cases the sum of two Boltzmann functions plus a residual component significantly improved the goodness of fit where a and b are the fractional amplitudes of the two functions and r is the residual component, i.e., (1  a  b). Fits of theoretical equations to the experimental data were performed using pCLAMP software (Clampfit) or Prism (Graphpad Software, San Diego, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Steady-state inactivation of total outward current. A typical family of outward current traces after various V_m (Fig. 1A) revealed distinct differences in inactivation pattern for peak and late outward current (I_peak and I_late, respectively). With a V_m of 140 mV (trace 1), the test current at +60 mV rapidly reached its peak value at the beginning of the clamp step (I_peak) and declined to 70% of I_peak toward the end of the clamp step (I_late), indicating that a large fraction of outward current did not inactivate during the test clamp step. In the voltage range negative to the resting potential the amplitude of the test pulse current became smaller, with less decrease in I_peak than in I_late causing an apparent increase in the transient component (compare traces 1 and 2 in Fig. 1). After two to three conditioning steps with little change in test current giving rise to a plateau, I_peak was strongly diminished by V_m between 60 and 30 mV (trace 3). The remainder of the current was inactivated by V_m up to 20 mV, and the residual current positive to this potential was resistant to any inactivation (trace 4).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Steady-state inactivation curves in a representative rat ventricular myocyte. A: superimposed current tracings elicited by stepping to test potential of +60 mV after conditioning clamp steps in range of 140 (trace 1) to +20 (trace 4) mV; traces 2 and 3 correspond to conditioning potentials (Vm) of 60 and 40 mV, respectively. Dashed line marks zero current level. Inset: clamp protocol. Increments of Vm were 10 mV except between 60 and 20 mV, where 5-mV steps were used. Ipeak, peak current; Ilate, late current. B: both Ipeak and Ilate were normalized to maximum peak current (I/Imax) and plotted as a function of Vm; data were fitted by sum of 2 Boltzmann functions (see METHODS; best line fit of data points from A). First phase of inactivation is half-maximum for Ipeak as well as for Ilate around 100 mV (left vertical line, top). However, second phase of inactivation is half-maximum around 45 mV for Ipeak (right vertical line, arrow, top), but around 35 mV for Ilate (right vertical line, arrow, bottom); note that transient part of component b is almost completely inactivated when sustained part just begins to be inactivated (trace 3 in A). Both Ipeak and Ilate show 3 phases: a, b, and r.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Correlation of amplitudes (in pA/pF) of outward current components. Ilate is shown as function of Ipeak for fractions a (A), b (B), and r (C). Note highly significant correlation between Ipeak and Ilate in a and r, respectively, but lack of correlation in b (Spearman's rank correlation). Cells isolated from cardiac apex (subepicardial, ), cardiac base (subendocardial, ), and whole left ventricular free wall (). rs, Correlation coefficient; n, no. of experiments.

Voltage dependence of activation of outward current components. The steady-state inactivation data presented so far allow us to distinguish a total of four outward current components, i.e., I_K, I_to, I_Kx, and I_ss. To obtain data on the activation kinetics and voltage dependence of the four current components, outward currents were activated by stepping to voltages in the range of 40 to +60 mV from the three V_m of 140, 70, and 20 mV (Fig. 3, A-C) followed by digital subtraction of current tracings. This procedure was aimed at further characterizing the individual current components (Fig. 3, D-E). Because channel availability should be at its maximum with a V_m of 140 mV (compare Fig. 1), the amplitude of activated outward current is large (Fig. 3A). Both I_peak and I_late are decreased after conditioning steps to 70 mV (Fig. 3B), where steady-state inactivation of I_K should be complete (Fig. 1; Table 1). Therefore, digital subtraction of these two sets of current tracings should result in the isolation of I_K (Fig. 3, D and F). Currents activated from a V_m of 20 mV should reflect activation of I_ss (Fig. 3, C and H). Hence, the difference between V_m of 20 and 70 mV should represent the sum of I_to and I_Kx (Fig. 3, E and G), which cannot be separated electrophysiologically because steady-state inactivation occurs in overlapping voltage ranges (Fig. 1; Table 1).

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Outward current activated from various Vm. A-C: superimposed current tracings elicited by stepping to potentials between 40 and +60 mV (see inset) after 2,000-ms conditioning clamp steps to 140 (A), 70 (B), and 20 (C) mV, respectively. Zero current level at horizontal bar. D and E: difference currents obtained by digital subtraction of tracings displayed in A-C: D = A  B, and E = B  C. Vertical calibration bar starts at 0 nA. F-H: voltage (V) dependence of current activation after digital subtraction of tracings recorded at Vm of 140 and 70 mV (F) and 70 and 20 mV (G) and of tracings measured at Vm of 20 mV (H). Nos. in parentheses indicate no. of experiments.

Sensitivity of outward current components to pharmacological tools. So far, electrophysiological evidence in support of four outward current components has been presented. In another approach to channel differentiation, we made use of several pharmacological tools that selectively block individual currents. For instance, 4-AP selectively blocks I_to (4, 12), TEA attenuates I_K (4, 35), and quinidine or clofilium reduces both current components (10, 26, 35). DTX is a potent blocker of a delayed-rectifier-like current flowing through the cloned Kv1.2 channel (13, 36). HpTx3 selectively blocks cloned and native Kv4.2 channels (32), whereas hanatoxin blocks both Kv4.2 and Kv2.1 channels, as shown in a Xenopus expression system (36). Because the selectivity and efficacy of these agents are usually maintained after expression of cloned channels in mammalian cell lines, the sensitivity profile can be used for channel identification (6, 22).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Heteropodatoxin-3 (HpTx3, 2 µM) shifts steady-state inactivation curve. A: superimposed current tracings elicited by stepping to test potential of +60 mV after conditioning clamp steps in range of 140 (trace 1) to +20 (trace 4) mV; traces 2 and 3 correspond to Vm of 60 and 20 mV, respectively. Dashed line marks zero current level. For clamp protocol see inset of Fig. 1. B: both Ipeak (top) and Ilate (bottom) were normalized to Imax and plotted as a function of Vm; data obtained under control conditions (n = 7 experiments) and after exposure to HpTx3 (2 µM) were fitted by sum of 2 Boltzmann functions (see METHODS; best line fit). Note that, in range of 60 to 0 mV, monophasic steady-state inactivation of Ipeak under control conditions becomes biphasic in presence of HpTx3 (arrows). Parameters of Boltzmann curve fitting for Ilate were V0.5,a 97 ± 3 mV, ka 9 ± 1 mV, a 15 ± 2%, V0.5,b 36 ± 2 mV, kb 6 ± 1 mV, b 9 ± 1%, and r 14 ± 2%, and they were unaffected by HpTx3; For Ipeak, parameters were V0.5,a 99 ± 5 mV, ka 12 ± 1 mV, a 15 ± 4%, V0.5,b 37 ± 1 mV, kb 4 ± 1 mV, b 68 ± 6 %, r 15 ± 3%; although a and r remained unaffected, HpTx3 shifted a portion of b resulting in V0.5,b1 41 ± 1 mV, kb1 6 ± 1 mV, b1 16 ± 3%, V0.5,b2 8 ± 1 mV, kb2 4 ± 1 mV, and b2 23 ± 2%.

View larger version (52K): [in this window] [in a new window]   Fig. 5.   Effects of pharmacological tools 4-aminopyridine (4-AP), tetraethylammonium (TEA), clofilium (Clof), quinidine (Quin), and dendrotoxin (DTX) on various components of outward current expressed in percentage of respective control values. Top: sustained TEA-sensitive current component IK; component a). Middle: transient 4-AP-sensitive current component (Ito; fraction b of Ipeak) and novel current component (IKx; fraction b of Ilate). Bottom: steady-state current component (Iss; component r). Concentrations used were 100 µM (n = 7 experiments) and 1 mM (n = 10) for 4-AP, 1 (n = 8), 3 (n = 7), and 10 (n = 8) mM for TEA, 3 (n = 7) and 30 (n = 11) µM for clofilium, 5 µM (n = 8) for quinidine, and 100 nM (n = 7) for DTX. Mean values ± SE of Ipeak (cross-hatched bars)- and Ilate (solid bars)-derived data are shown for test potential of +60 mV. * Statistically significant differences between Ipeak- and Ilate-derived data (P < 0.05, Student's t-test for paired data). # Statistically significant differences vs. control level (= 100%) (calculated with 1-sample t-test; P < 0.05).

Ion selectivity of outward current components. Ion selectivity is another criterion to distinguish between different ion channels. Other ions than K⁺ could contribute as charge carriers in generating the four current components. In particular, a nonselective cation current carried by K⁺, Na⁺, and Ca²⁺ or an anion background current carried by Cl could be involved. To test for ion selectivity, the intracellular ion concentration was varied by substituting K⁺ in the pipette solution with either Cs⁺ or TEA, both of which permeate poorly through K⁺ channels, or by lowering extracellular Cl from 163 to 13 mM by substituting sodium methanesulfonate for NaCl. The results of the respective experiments are summarized in Fig. 6. Replacement of K⁺ by Cs⁺ in the pipette solution significantly depressed I_K, abolished I_Kx (b of I_late), and reduced the amplitude of I_to (b of I_peak) and I_ss to <50%. The latter current components were further decreased when TEA was present in the pipette solution. With 90% of extracellular Cl replaced by the membrane-impermeant methanesulfonate, only I_ss was reduced to 70%, whereas the other current components were not affected. These results suggest that the majority of outward current was carried by K⁺ and that I_ss consisted of two separate currents, one of which appeared to be carried by K⁺ and the other of which was most likely a Cl current.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Alteration of ionic composition of pipette or bath solution affects size of current components. K+ in electrode solution (140 mM, control = 100%; n = 11 experiments) was completely replaced by Cs+ ([Cs+]i, n = 5) or by TEA ([TEA+]i, n = 3); mean dialysis time was 7 ± 1 (Cs+) and 9 ± 1 (TEA) min after transition into whole cell mode. Current amplitudes of [Cs+]i and [TEA]i groups were normalized to average maximal current of [K+]i group. [Cl] in bath solution ([Cl]o) was lowered from 163 (control) to 13 (n = 6) mM by substituting sodium methanesulfonate for NaCl. Test potential +60 mV; layout and statistics as in Fig. 5.

Transmural distribution of outward current components. Within the ventricular wall, I_to was found to be more prominent in subepicardial than in subendocardial myocytes of rat ventricle (14), and a similar distribution has been reported for K⁺ channels at the mRNA and protein levels (7, 16). Myocytes of different transmural location can be obtained from rat hearts by isolating myocytes separately from the apex and the base, yielding subepicardial and subendocardial cells, respectively (14). Using this approach, we consistently observed that subepicardial myocytes possessed a large rapidly inactivating transient outward current, whereas subendocardial myocytes isolated from the base of the heart were characterized by a small I_to component (data not shown).

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Absolute amplitude (in pA/pF) of outward current components Ito (A), IKx (B), IK (C), and Iss (D) as function of relative contribution of Ito to total outward current. Data originate from myocytes isolated from whole left ventricular free wall () or isolated separately from apex () and base () of heart. Spearman's rank test yielded correlation coefficients rs and probability values indicated. In B and C, no. of experiments (n) deviates from those in A and D because steady-state inactivation curves with very small IK or IKx components could not be reliably fitted with sum of 2 Boltzmann functions.

Outward current component I_K and repolarization of action potential. The outward current components I_to, I_Kx, and I_ss should contribute to the shape of the action potential, as judged by the potential range of their availability. For component I_K, however, V_0.5 was 93 mV (see Table 1), and therefore this current component should be largely inactivated at normal RMP. Hence, its role for the action potential is less obvious than with the other current components. Here we tested whether increasing the availability of I_K by hyperpolarizing the membrane could influence the shape of the action potential. Action potentials recorded from subendocardial myocytes are much longer than those from subepicardial myocytes because of their profound difference in I_to (Fig. 8A). Action potentials measured at hyperpolarized potentials were markedly shortened in duration, and this effect was significantly greater in subendocardial than in subepicardial cells (Fig. 8B). This observation was consistent with the theoretically expected increase in I_K availability under hyperpolarizing conditions. Because I_K was half-inactivated at 93 mV (Table 1), its availability at a normal resting potential of 70 mV amounted to 8%. Average hyperpolarization by 12 mV should have increased the availability to 24%, and this additional repolarizing current strongly reduced APD in subendocardial myocytes. The much lesser effect on APD in subepicardial cells was probably caused by the larger repolarizing force of I_to. Conversely, the blocking effect of TEA (10 mM) on outward currents did not produce any prolongation in APD in subepicardial myocytes (data not shown), whereas action potentials in subendocardial cells were markedly prolonged at both normal and hyperpolarized resting potentials (Fig. 8C). These data suggest that I_K may contribute to repolarization at least in subendocardial myocytes.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   A: superimposed representative action potentials from a subendocardial (Endo) and a subepicardial (Epi) myocyte. Dotted line at 0 mV, calibration as indicated. B: mean values ± SE from action potential measurements in Endo (n = 5) and Epi (n = 4) myocytes with normal resting membrane potential (N) or when a hyperpolarizing current was injected (H). Membrane potentials were 69.7 ± 1.5 (N) and 82.1 ± 2.6 (H) mV in Endo cells and 72.9 ± 1.7 (N) and 85.2 ± 1.9 (H) mV in Epi cells; average hyperpolarization amounted to 12.3 ± 1.5 (Endo) and 12.5 ± 1.4 (Epi) mV. APD20, APD50, APD90, action potential durations at 20, 50, 90% repolarization. Nos. in parentheses indicate no. of experiments. C: superimposed action potentials measured at normal and hyperpolarized resting membrane potential (RMP) in a representative Endo myocyte. Con, control; TEA, 10 mM TEA. Horizontal dotted line at 0 mV, calibration as indicated. Similar results were obtained in 3 other myocytes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Outward current in rat ventricular myocytes consists of at least four different components that are distinguished on the basis of time course, potential range of steady-state inactivation, sensitivity to pharmacological blockers, and gradient of amplitude within the ventricular wall. In addition to I_to, two delayed rectifier-like currents (I_K, I_Kx) and at least one noninactivating background component (I_ss) were identified.

Dissection of outward current components. In native rat ventricular myocytes, two major outward current components are regularly detected, i.e., the transient, 4-AP-sensitive K⁺ current I_to and the sustained TEA-sensitive K⁺ current I_K (4, 6, 14). For I_to, half-maximum steady-state inactivation (V_0.5) is found at potentials between 29 and 46 mV (4, 43). This difference in V_0.5 values from the various studies may be caused in part by divalent cations (i.e., Cd²⁺ or Co²⁺) that are used to block Ca²⁺ current and are known to shift steady-state inactivation curves to the right (1). The steady-state inactivation of I_K has a more shallow potential dependence than I_to; V_0.5 values are reported between 77 and 114 mV (4, 11). Occasionally, a noninactivating residual outward current is observed that persists in the presence of 4-AP and TEA and contributes 10-30% to peak outward current (4, 19, 35, 40, 42).

Relation to cloned voltage-dependent K⁺ channels. In rat ventricle, a multitude of depolarization-activated K⁺ channels have been identified at the mRNA level, whereas only two current phenotypes, I_to and I_K, have been distinguished (7, 13, 16, 30). Heterologous expression of Kv channels allows their pharmacological profiling. Our data on fraction b of I_peak (transient time course, inactivation kinetics, pharmacological profile, transmural gradient) are consistent with the idea of its identity to I_to and confirm the role of proteins of the Kv4 family in generating I_to in rat ventricle (compare HpTx3 data). The kinetic properties of component a resemble those of the delayed rectifier I_K. However, the present data and our indirect experimental approach do not allow a definite conclusion about the nature of the Kv channel responsible for I_K. In particular, component a, i.e., I_K, is a sustained current without transmural gradient and is sensitive to block by TEA and hanatoxin (blocker of Kv2.1 and Kv4.2; Ref. 36) but is insensitive to 4-AP, dendrotoxin (blocker of Kv1.2; Ref. 13), and HpTx3 (blocker of Kv4.2; Ref. 32). This pattern could give rise to the hypothesis that Kv2.1 might underlie I_K. Finally, although the current components I_Kx and I_ss were unaffected by either of the toxins used, this lack of effect cannot be interpreted in terms of absence of the respective Kv gene products (particularly Kv1.2). Moreover, the reason for this finding is unclear and requires further investigation. In any case, Kv channel gene products can only be related to native currents with great caution, because of the inherent differences in heteromultimeric composition and accessory subunits of K⁺ channels between expression systems and native myocytes (31).