Introduction

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Calmodulin-dependent protein kinase II (CamKII) is a multifunctional cytosolic enzyme that plays an important role in regulating cardiac and smooth muscle Ca²⁺ homeostasis and contractility. This enzyme is stimulated by the binding of the Ca²⁺-calmodulin complex after elevation of free intracellular calcium concentration ([Ca²⁺]_i) and by a series of autophosphorylation steps (Singer et al., 1996). CamKII is known to play a major role in the facilitation of Ca²⁺ current during enhanced repetitive stimulations after a period of rest in cardiac and smooth muscle cells (McCarron et al., 1992; Yuan and Bers, 1994) and to increase the activity of the cardiac and skeletal muscle sarcoplasmic reticulum Ca²⁺-ATPase (Hawkins et al., 1994; Li et al., 1997) and Ca²⁺ release channel (Takasago et al., 1991; Li et al., 1997).

Four separate classes of K⁺ channels have so far been identified in vascular smooth muscle cells. These include large-conductance Ca²⁺-dependent K⁺ channels (K_Ca), voltage-dependent or delayed rectifier K⁺ channels (K_V), ATP-sensitive K⁺ channels, and inwardly rectifying K⁺ channels (Nelson and Quayle, 1995). Among these, it is well accepted that the 4-aminopyridine (4-AP)-sensitive K_V current plays a prime function in regulating resting membrane potential and vascular tone, especially at low or resting levels of [Ca²⁺]_i (Leblanc et al., 1994; Nelson and Quayle, 1995). It has been recently reported that K_V, which may be the result of several components and at least two distinct molecular entities, namely K_V1.5 (Overturf et al., 1994) and K_V1.2 (Hart et al., 1993), can be up- and down-regulated by phosphorylation mediated by protein kinase A (Aiello et al., 1995) and C (Clement-Chomienne et al., 1996), respectively. It is unknown at the present time whether K_V channels are modulated by phosphorylation involving CamKII.

Several novel organic compounds have recently been developed to study the biochemical and functional properties of CamKII in different biological preparations. Among these, KN-93, a methoxybenzenesulfonamide compound, has been reported to be a highly specific inhibitor of CamKII in the brain, with little or no influence on the activity of PKA, PKC, and other important protein kinases (Sumi et al., 1991). As for KN-62, another CamKII inhibitor, KN-93 was reportedly shown to bind to the Ca²⁺-calmodulin domain of CamKII and thus prevent the stimulation of this enzyme (Tokumitsu et al., 1990; Sumi et al., 1991). In view of the important role of K_V in controlling the resting membrane potential and vascular tone in vascular myocytes (Nelson and Quayle, 1995), our main objective was to investigate the effects of KN-93, KN-92, an analog of KN-93 bearing no influence on CamKII, and those of another CamKII blocker, KN-62, on macroscopic K_V currents recorded in freshly dissociated rabbit portal vein smooth muscle cells at 35°C. We provide evidence that the two widely used inhibitors of CamKII, KN-93 and KN-62, both inhibit whole-cell K_V with a potency well within the range of their respective effects on CamKII. These inhibitory actions do not appear to be mediated by CamKII, because they could still be observed with high intracellular Ca²⁺ buffering or mimicked by the inactive analog of KN-93, KN-92. These results have been presented in preliminary format (Leblanc and Chartier, 1998).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Single-Cell Preparation. Isolated vascular smooth muscle cells were enzymatically dispersed from the rabbit portal vein as previously described (Leblanc and Leung, 1995). In brief, albino rabbits of either sex weighing 2.5 to 3 kg were sacrificed by an overdose of pentobarbital sodium injected through the ear vein. After exsanguination, the portal vein was removed and immediately immersed in cold (4°C) and well oxygenated physiological salt solution (PSS) containing: 120 mM NaCl, 15 mM NaHCO₃, 4.2 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 0.01 mM CaCl₂, and 5.5 mM dextrose (pH = 7.4 when bubbled with 95% O₂-5% CO₂)_. The portal vein was first pinned down at the bottom of a Petri dish layered with Sylgard and containing well oxygenated PSS. Under the view of a stereomicroscope (model SMZ-1B; Nikon Corp., Tokyo, Japan), the vessel was cut open along the longitudinal axis and was freed of blood, fat, and connective tissue by using fine dissecting tools. After dissection, the vein was cut into small pieces (2 × 2 mm), which were transferred into a small beaker containing 10 ml of a solution similar to that described above except that it was nominally Ca²⁺ free (no added CaCl₂), and it contained the Ca²⁺ chelating agent EGTA (0.1 mM). After 30 min of incubation at room temperature, the tissue pieces were transferred in the PSS solution containing the following enzymes: collagenase (0.2 mg/ml, type 1A; Sigma Chemical Co., St. Louis, MO) and protease (50 µg/ml, type XXVII; Sigma Chemical Co.). This incubation was allowed to proceed at 35°C for 20 to 25 min, based on the number of cells released during trituration of a few pieces in low Ca²⁺ PSS by using a Pasteur pipette. Once a large number of cells was observed under microscopic examination, all pieces were rinsed several times in enzyme-free PSS and triturated to release single spindle-shaped smooth muscle cells. The supernatant containing the single cells was collected and kept in the cold (4°C) until use. All experiments were carried out within 4 to 8 h after isolation.

Electrophysiology. Calcium-tolerant portal vein myocytes were either current or voltage clamped using the standard or perforated (nystatin) variant of the whole-cell patch clamp technique (Hamill et al., 1981). Large-diameter micropipettes were pulled by using a two-stage micropipette puller (model PP-83; Narishige Scientific Laboratory, Tokyo, Japan) and polished using a microforge (model FP-83; Narishige Scientific Laboratory). With tip diameters of about 1 µm, the pipette resistances were in the range of 2 to 4 M when filled with the internal solutions described below. Voltage or current clamp protocols were computer driven using pClamp software (Version 5.5.1) and an Axopatch 200A integrating patch amplifier (Axon Instruments, Inc., Foster City, CA). Pipette and stray capacitances and series resistance were compensated for in all voltage clamp experiments. Membrane current was low-pass filtered at 1 or 2 kHz (four-pole bessel filter) before being acquired at a sampling rate of 2 or 5 kHz by using a PC-486 computer interfaced with a 12-bit analog-to-digital acquisition board (TL-125; Axon Instruments, Inc.). The resting membrane potential was measured in the current clamp mode by using the perforated patch clamp technique. The output voltage signal was filtered at 1 kHz and converted to digital format (sampled at 100 Hz) by using the same acquisition system and the Axotape software (Version 2.0; Axon Instruments, Inc.). Once digitized, the data were temporarily stored on the computer hard disk for later analysis (pClamp Version 6.0 or Axotape Version 2.0; Axon Instruments, Inc.) and display (Hewlett-Packard LaserJet series III).

Solutions. With the exception of L-type Ca²⁺ currents that were recorded at room temperature (Fig. 1), all other experiments were carried out at 35°C. In voltage clamp experiments designed to measure K⁺ currents (Figs. 2-8) and all current clamp protocols (Fig. 9), the solution used to superfuse the myocytes had the following composition: 130 mM NaCl, 10 mM NaHCO₃, 4.2 mM KCl, 1.2 mM KH₂PO₄, 0.5 mM MgCl₂, 1.8 mM CaCl₂, 5.5 mM dextrose, and 5.0 mM HEPES-NaOH (pH 7.35). Except for the data presented in Figs. 1, 2, and 9, all other experiments were carried out in the presence of 1 µM nifedipine to inhibit L-type Ca²⁺ channels. To record L-type Ca²⁺ channels in isolation (Fig. 1), the same solution was used except that KCl and KH₂PO₄ were replaced by 5.4 mM tetraethylammonium chloride (TEA).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Evidence for functional CamKII activity in rabbit portal vein myocytes. A, selected L-type Ca2+ current (ICa) tracings recorded with the perforated patch technique during a train of 100-ms steps to 0 mV imposed at a rate of 0.5 Hz from HP of 70 mV, as depicted. Currents elicited during the first pulse for each condition were used to scale all currents to the same amplitude for comparison. Each train was preceded by a 2-min rest period while the myocyte was maintained at HP. For each condition, pulses 1, 4, and 30 are shown. Superimposed traces obtained in the absence (Control, leftward traces) and after 7 min of incubation with 1 µM KN-62. B, effects of KN-62 on the frequency dependence of ICa. For each condition, the magnitude of ICa currents recorded during a train of 32 pulses to 0 mV from HP = 70 mV (0.5 Hz) was normalized against that elicited during the first pulse. The graphs represent mean data of normalized currents from six experiments similar to that of A. Lines passing through the data points are least-square double-exponential fits of the following form:  + C, where A1 and A2 are the amplitudes of the currents for the exponential components, 1 and 2 are the time constants for the two kinetic processes, and C is a constant. The following parameters were derived for each set of data: Control: A1, .11; 1, 2.29 pulses, A2, .15; 2, 21.7 pulses, and C, .79; KN-62: A1, .38; 1, 1.93 pulse, A2, .36; 2, 52.2 pulses, and C, .43. All comparisons between Control and KN-62 data points revealed significant differences with at least p < .05 (paired t test).

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Effects of KN-93 on K+ currents recorded from portal vein smooth muscle cells dialyzed with low EGTA pipette solution. A, family of whole-cell K+ currents recorded in control (Control, top left), presence of 5 µM KN-93 (top right) and after return to drug-free solution (Washout, bottom left). The currents were evoked by the protocol shown at the bottom right. B, I-V relationships for peak (left) and late (right) currents as in A in Control (, ), in presence of 5 µM KN-93 (, ), and after Washout (). Each data point is a mean ± S.E.M. (n = 6). All comparisons for peak and late currents between Control and KN-93 revealed significant differences at potentials  20 mV with at least p < .05 (paired t test). Pipette solution contained 0.1 mM EGTA, as indicated.

Statistical Analysis. Except where original tracings of membrane currents or resting membrane potential are displayed, all data are reported as means ± S.E.M. Where appropriate, a paired Student's t test was used to determine the difference between two groups. Comparisons between more than two groups were evaluated by a one-way ANOVA. Least-squares fits to mean data points by using a double-exponential formalism (Fig. 1) or the appropriate forms of the Boltzmann equation (Fig. 6) were performed by the Origin software (Version 4.1; Microcal Softwares, Inc., Northampton, MA). A probability p < .05 was accepted as the level of significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Activation of CamKII by elevation of [Ca²⁺]_i has been demonstrated to up-regulate the activity of L-type Ca²⁺ current (I_Ca) in smooth (McCarron et al., 1992) and cardiac (Yuan and Bers, 1994) muscle cells. We took advantage of this relationship to provide evidence for functional CamKII activity in our preparation. Figure 1 shows the results of experiments illustrating the stimulation-dependence of I_Ca elicited by a train of 32 pulses to 0 mV from holding potential (HP) of 70 mV (0.5 Hz), in the absence (Control) and presence of 1 µM KN-62, a CamKII inhibitor. The more potent blocker of CamKII, KN-93, could not be used, because it inhibited I_Ca by more than 50%, possibly because of a direct effect on the channel. Cell exposure to 1 µM KN-62 for 5 to 7 min did not significantly influence I_Ca as monitored by repetitive depolarizations to 0 mV from HP = 70 mV applied every 20 to 30 s (n = 6); the amplitude of I_Ca in the absence and presence of KN-62 was 83 ± 28 and 76 ± 22 pA, respectively (p = .55). Each train of pulses was preceded by a 2-min rest period at the holding potential. Figure 1A shows selected I_Ca currents at different times during each train. In this experiment, control I_Ca currents evoked by the first and fourth pulses were similar, whereas that triggered by the 30th pulse displayed a slightly reduced amplitude. In contrast, I_Ca declined more quickly and to a lower level in the presence of KN-62. Figure 1B shows plots of mean normalized currents for the stimulation-dependent changes of I_Ca in control and after incubation with 1 µM KN-62. In control condition, the decline of Ca²⁺ current during the onset of stimulation can be explained, at least in part, by incomplete recovery from inactivation. It can be noticed that the current elicited during the second^. pulse (arrow) was not significantly different from that induced during the first pulse (p = .55). The rate of decline of I_Ca was enhanced and the steady-state level reached at the end of the train was lower in the presence of the CamKII inhibitor. In contrast, in the presence of KN-92 (data not shown), an analog of KN-93 that does not influence CamKII, the time course of decline of I_Ca during a train paralleled that seen in the absence of drug (n = 4). These results are compatible with the idea that Ca²⁺ accumulation during repetitive opening of I_Ca exerts rapid positive feedback regulation of I_Ca likely by the involvement of a phosphorylating step mediated by CamKII, as proposed for cardiac and smooth muscle cells (McCarron et al., 1992; Yuan and Bers, 1994).

We then examined the effects on K⁺ currents measured in cells dialyzed with low EGTA pipette solution of a concentration of KN-93 known to produce a potent inhibition of CamKII in the brain (K_i = 370 nM; Sumi et al., 1991). Figure 2A shows typical families of membrane currents elicited by the protocol shown at the lower right, in control (top left), after incubation with 5 µM KN-93 (top right), and after washout (bottom left). In the control condition, the time-dependent outward current elicited by such a protocol is composed of two dominant K⁺ currents: 1) a putative voltage-dependent K⁺ current (K_V) that displays slow inactivation at positive potentials and is sensitive to block by 4-AP (Beech and Bolton, 1989a; Hume and Leblanc, 1989; Miller et al., 1993), and a Ca²⁺-dependent K⁺ current (K_Ca) carried by large conductance maxi-K_Ca channels that are sensitive to low doses of TEA (1 mM), and to charybdotoxin (Beech and Bolton, 1989a) and iberiotoxin (Morales et al., 1996). As evident, KN-93 produced a potent inhibition of peak and late outward K⁺ currents at all potentials in the range of 20 to +60 mV. These effects were accompanied by marked increases in the rate of activation and inactivation. The KN-induced changes in outward K⁺ currents were partially reversible upon washout.

Figure 2B reports mean current-voltage (I-V) relationships for peak current (left graph) and current measured at the end of the 250-ms steps (right graph) in six experiments similar to that shown in Fig. 2A. KN-93 reduced peak and late currents, but the latter was more sensitive to block. For example, at +40 mV, KN-93 decreased peak (control: 53.0 ± 7.9 pA/pF; KN-93: 27.8 ± 3.83 pA/pF) and late (control: 32.8 ± 6.2 pA/pF; KN-93: 4.8 ± 0.9 pA/pF) by 48% and 85%, respectively. Consistent with the data described in Fig. 2A, washout of KN-93 led to incomplete recovery of peak (51% at +40 mV) and late (39% at +40 mV) outward K⁺ current.

Because the above experiments did not allow us to distinguish the effects of KN-93 on K_V and K_Ca, we carried out another series of experiments in conditions set to minimize the activity of K_Ca and record K_V in isolation. The myocytes were dialyzed with 10 mM EGTA to buffer intracellular Ca²⁺ (<1 nM) and superfused with 1 µM nifedipine to block L-type Ca²⁺ channels. Figure 3A illustrates the results of one typical experiment in which the effects of KN-93 were tested on whole-cell K_V currents elicited by the voltage clamp shown at the lower right-hand side. Families of K⁺ currents (250 ms steps from 60 mV), which mainly consisted of transient voltage-dependent K⁺ currents (K_V), were recorded in the absence of drug (Control), after 6 min of incubation with 5 µM KN-93, and 8 min after return to control solution (Washout). As for cells dialyzed with low EGTA, KN-93 inhibited K_V at all step potentials and caused similar changes in activation and inactivation kinetics; both effects were partially reversible on drug removal.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of KN-93 on the voltage-dependent K+ current. A, typical family of whole-cell voltage-dependent K+ currents recorded in absence of drug (Control), 6 min after incubation with 5 µM KN-93, and 8 min after washout. The current was elicited by the protocol shown at the lower right-hand side. B, average I-V relationships for peak (, ) and late (, ) currents in absence (Control, , ) and in the presence of 5 µM KN-93 (, ). Each data point is a mean ± S.E.M. (n = 4). As for the data presented in Fig. 2B, all comparisons for peak and late currents between Control and KN-93 revealed significant differences at potentials 20 mV with at least p < .05 (paired t test). Myocytes were dialyzed with 10 mM EGTA and incubated in the presence of 1 µM nifedipine.

The graph depicted in Fig. 3B reports the average data for the I-V relationships of peak (squares) and late (circles) K_V currents measured in the absence (Control, filled symbols) and presence (empty symbols) of 5 µM KN-93. Because K_Ca activates in a time-dependent manner but does not inactivate, its contribution to total current should be greater at the end of a depolarizing step. Consistent with this proposal, the current at the end of steps was suppressed to a greater extent than peak current by enhanced intracellular Ca²⁺ buffering with elevated EGTA concentration in the pipette solution (compare Figs. 2B, 3B, and 7, A and B). At +40 mV, peak current was 53.0 ± 7.9 and 34.2 ± 5.2 pA/pF with low- and high-EGTA pipette solutions, respectively, a difference near the limit of significance (p = .123); late current was 32.8 ± 6.2 and 15.8 ± 3.2 pA/pF (p = .011) with low- and high-EGTA pipette solutions, respectively. KN-93 blocked peak and late K_V current but with more prominent effects on the latter. The magnitude of block of the peak and late K_V current components by KN-93 displayed little voltage dependence for step potentials ranging from 20 to +60 mV (for peak and late current components, percentage block ranged from 50 to 54% and from 87 to 93%, respectively). The percentage of block produced by KN-93 in the presence of nifedipine and 10 mM EGTA was similar to that measured in control conditions (Fig. 2) for peak (+40 mV: 48 versus 54% for low and high EGTA, respectively) and late (+40 mV: 85 versus 92% for low and high EGTA, respectively).

Separate experiments were performed to quantify the potency of KN-induced block of K_V. Figure 4A shows superimposed tracings of K_V currents elicited by the voltage clamp protocol shown at the bottom. The currents were recorded in the same cell in the absence (Control) and presence of 0.1, 0.5, 1, and 10 µM KN-93, as depicted. As shown above, KN-93 inhibited both peak and late currents, but it was more potent on late current. It is also evident that KN-93 produced an apparent increase in the rate of development of inactivation, an effect that was also dose dependent.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent inhibition of KV by KN-93. A, whole-cell KV currents elicited by the protocol shown at the bottom were all recorded in the same cell in absence (Control, ) and presence of 0.1, 0.5, 1, and 10 µM KN-93, as depicted by the various open symbols. B, concentration-response relationships expressed in percentage of block of KV for peak () and late () outward currents recorded at +60 mV. Each data point is a mean ± S.E.M. (n = 6). The two smooth lines for the block of peak and late KV currents by KN-93 were obtained by nonlinear least-squares fitting of the mean data points to a Logistic function of the following form: Y = 100/{1 + (IC50/[KN-93])p}, where Y represents percentage block, [KN-93] is the concentration of KN-93 in µM, IC50 is the concentration (in micromolar concentrations) of KN-93 producing 50% inhibition of the current, and p is the power factor. For peak current (), the IC50 (as labeled) and p values are 2.9 µM and .64, respectively, and for late current (), the IC50 (as labeled) and p parameters are .27 µM and 1.301, respectively. Myocytes were dialyzed with 10 mM EGTA and exposed to 1 µM nifedipine.

Figure 4B shows the concentration-response relationships for KN-93-induced block of K_V for peak (filled squares) and late (empty circles) currents recorded at +60 mV. The two smooth lines are nonlinear least-squares fits to a Logistic function. As evident from the IC₅₀ values indicated, KN-93 was about 10 times more potent at inhibiting late versus peak currents. These results show that KN-93 inhibits K_V in a dose-dependent manner.

Figure 5 displays graphs illustrating the voltage dependence of the fast (_fast, A) and slow (_slow, B) time constants of inactivation in the absence (Control, filled squares) and presence (empty circles) of 5 µM KN-93. In the absence of drug, both _fast and _slow decreased from 10 to +10 mV but displayed little, if any, voltage dependence at potentials +20 mV. KN-93 produced an 2- to 3-fold reduction of _fast and _slow.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effects of KN-93 on inactivation kinetics of KV (A and B). Voltage dependence of the fast (fast, A) and slow (slow, B) time constants of inactivation in control () and in presence of 5 µM KN-93 (). For A and B, all comparisons revealed significant differences between time constants measured in Control and KN-93 at all potentials with p < .05 (paired t test). Myocytes were dialyzed with 10 mM EGTA and exposed to 1 µM nifedipine.

To obtain information on the possible mechanism of block of K_V by KN-93, we also examined its effects on the steady-state activation and inactivation properties of K_V. For these experiments, a lower concentration of KN-93 (1 instead of 5 µM) was tested to record larger K_V currents to facilitate the analysis. A double-pulse protocol was used to construct the steady-state activation curves shown in Fig. 6A. From holding potential of 70 mV, 5-ms steps (P1) from 50 to +30 mV were applied in 10-mV increments at a frequency of 0.2 Hz to activate K_V; each of these steps was followed by a constant pulse (P2) to 35 mV to record outward tail current. The amplitude of each tail current recorded during P2 was normalized against the maximum tail-current amplitude and plotted as a function of the voltage step during P1. KN-93 shifted the activation curve by 7 mV (V_0.5: from 12 ± 2 to 19 ± 3 mV, p < .05) and enhanced the steepness of the relationship (slope factor k: from 8.8 ± 0.4 to 6.3 ± 0.5 mV, P< .01).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effects of KN-93 on the steady-state activation and inactivation properties of KV. A, effects of 1 µM KN-93 on the steady-state activation curve of KV. From holding of 70 mV, 5-ms steps (P1) from 50 to +30 mV (10-mV increments) at a frequency of 0.2 Hz were used to activate KV. P1 was followed by a second constant pulse to 35 mV (P2) to record outward deactivating tail current. The amplitude of outward tail current was normalized against the maximum tail current amplitude and plotted as a function of the voltage step during P1. Each data point is a mean ± S.E.M. of normalized tail current (n = 5). The two lines are least-squares Boltzmann fits to the data using the following formulation: Y = {1 + exp[(V0.5  V)/k]}1, where Y is the value of the steady-state activation gating variable, V0.5 is the voltage at which KV is 50% activated, and k is the slope factor; in control: V0.5 = 11.9 and k = 9.2 mV; with KN-93: V0.5 = 19.0 mV, and k = 7.5 mV. B, effects of 1 µM KN-93 on the relative availability of KV. From holding potential of 70 mV, a 10-s variable conditioning step (P1) from 100 to 10 mV (10-mV increments) was applied at a rate of 0.1 Hz. After each of these pulses, a constant 5-ms pulse to 100 mV (P2) preceded a 45-ms step to +60 mV (P3) to record outward current. Amplitude of peak outward current recorded during P3 was normalized against the maximum outward current amplitude and plotted as function of the voltage step in P1. Each data point is a mean ± S.E.M. of normalized current (n = 5). The two lines are least-squares Boltzmann fits to the data using the following formulation: Y = {1 + exp[(V  V0.5)/k]}1, where Y is the value of the steady-state inactivation gating variable, V0.5 is the voltage at which KV is 50% inactivated, and k is the slope factor; in control: V0.5 = 46.2 mV, and k = 9.6 mV; with KN-93: V0.5 = 51.8 mV, and k = 10.2 mV. Myocytes were dialyzed with 10 mM EGTA and exposed to 1 µM nifedipine.

A three-step protocol was used to construct the steady-state inactivation curve of K_V in control and presence of 1 µM KN-93. From HP = 70 mV, the membrane was first conditioned for 10 s to potentials varying from 100 to +10 mV (P1) applied in 10-mV increments at a rate of 0.1 Hz; this was followed by a constant 5-ms step to 100 mV (P2), itself followed by a 45-ms step to +60 mV (P3) to record K_V. The amplitude of K_V during P3 for each conditioning voltage step was normalized against the maximum amplitude of K_V and plotted against P1 voltage. KN-93 shifted the steady-state inactivation curve by 6 mV (V_0.5: from 45 ± 2 mV to 51 ± 3, p < .05) with little effect on the slope of the relationship (slope factor k: from 9.3 ± 1.1 to 9.4 ± 1.3 mV, p = .86).

We also explored whether the effects on K_V reported above are unique to KN-93. Fig. 7 reports the effects of another blocker of CamKII, KN-62, and of KN-92, the structural analog of KN-93. Figure 7A shows sample recordings of whole-cell currents elicited by the protocol shown at the bottom and obtained in control condition and after exposure to 5 µM KN-62. Although less potent than KN-93, KN-62 also inhibits K_V in a manner similar to that produced by KN-93: 1) the current at the end of the step was more sensitive to block by KN-62, and 2) the kinetics of inactivation were accelerated in the presence of the drug. To the right of Fig. 7A are depicted the mean I-V relationships for peak (squares) and late (circles) currents recorded in response to a protocol identical with those described in Figs. 2 and 3, in the absence (Control) and presence of 5 µM KN-62. On average, KN-62 did not significantly reduce peak current, although a small tendency was noticeable (10% at +60 mV). However, the CamKII inhibitor produced 40% inhibition of late current at +60 mV.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   The putative CamKII inhibitor KN-62 and KN-92, a structural analog of KN-93, also inhibit KV. (A, left) Sample traces of KV currents evoked by 250-ms steps to +60 mV in absence (Control, ) and in presence of 5 µM KN-62 (). Right, mean I-V relationships (n = 4) for peak (, ) and late (, ) outward currents elicited by protocol as in Figs. 2 and 3, in Control (, ) and in the presence of 5 µM KN-62 (, ). All comparisons for late currents between Control and KN-62 revealed significant differences at potentials 0 mV with at least p < .05 (paired t test). Comparisons for peak current indicated no significant difference at all potentials examined. (B, left) Sample traces of KV currents evoked by 250-ms steps to +60 mV in absence (Control, ) and in presence of 5 µM KN-92 (). Right, mean I-V relationships (n = 9) for peak (, ) and late (, ) currents elicited by protocol as in A, in absence (Control, , ) and presence of 5 µM KN-92 (, ). All comparisons for peak and late currents between Control and KN-92 revealed significant differences at potentials 0 mV with at least p < .05 (paired t test). C, bar graph reporting the relative potency of block by KN-93 (expressed as percentage of block) of peak and late KV measured at +60 mV. Myocytes were dialyzed with 10 mM EGTA and exposed to 1 µM nifedipine.

Figure 7B shows that 5 µM KN-92 produced a more potent inhibition of K_V than KN-62. The effects of KN-92 on inactivation kinetics were also more prominent than those caused by KN-62. Figure 7C shows a bar graph comparing the potency of inhibition of K_V at +60 mV by KN-93, KN-62, and KN-92 at a concentration of 5 µM. These results indicate a similar profile of inhibition of peak and late currents with the following order of potency: KN-93 > KN-92 > KN-62. In summary, KN-62 inhibits and affects the kinetics of K_V in a manner similar to KN-93, although less potent than the latter at equivalent concentrations.

Although it appears unlikely that KN-93 and KN-62 influenced K_V through an inhibitory effect of CamKII in myocytes dialyzed with high EGTA, we nevertheless further tested this hypothesis by examining the effects of cell dialysis with a synthetic peptide fragment (281-301) that inhibits CamKII by interacting with the calmodulin-binding domain of CamKII (IC₅₀ = 2 µM). Over a 15-min period, cell dialysis with a pipette solution containing 10 µM of the CamKII peptide inhibitor produced little, if any, effect on the amplitude and kinetics of K_V (Fig. 8A, upper and lower left families of currents). Under these conditions, cell exposure to 5 µM KN-93 for 5 min decreased K_V in a manner consistent with that described previously in Figs. 2, 3, and 7 (Fig. 8A, bottom right traces). Figure 8B reports mean I-V relationships for peak (left graph) and late (right graph) K_V currents recorded after 3 and 15 min of cell dialysis and after a subsequent exposure and washout of KN-93. The KN-93-induced block of K_V does not appear to be influenced by the presence of the inhibitor.

View larger version (35K): [in this window] [in a new window]   Fig. 8.   Effects of KN-93 on KV in the presence of a peptide inhibitor of CamKII. A, families of whole-cell KV currents recorded after 3 min (upper left), 10 min (upper right), and 15 min after membrane rupture. The cell was dialyzed with 10 mM EGTA and 10 µM of the synthetic peptide inhibitor of CamKII (fragment 281-301) and incubated with 1 µM nifedipine. The voltage clamp protocol is displayed at the lower left-hand side. The currents at the bottom right-hand side were obtained after 5-min exposure to 5 µM KN-93 or 20 min of cell dialysis. B, mean I-V relationships for peak (left graph) and late (right graph) KV currents derived from four experiments similar to that described in A. Pooled data for currents measured after 3 min () and 15 min () of cell dialysis and after exposure to 5 µM KN-93 (, 20 min of cell dialysis) and washout of the drug (, 25 min of cell dialysis). The data at 10 min of cell dialysis are not shown for the sake of clarity. All comparisons between 3 and 15 min revealed no statistically significant difference. All other comparisons showed significant differences for currents elicited by steps 10 mV.

Because of the known important function of K_V in regulating resting membrane potential (RMP) in vascular smooth muscle cells, our data would predict that KN-93 may induce membrane depolarization. We directly tested this hypothesis by examining the effects of KN-93 on RMP of portal vein myocytes measured in current clamp mode with the perforated patch clamp technique. Figure 9A shows a sample trace of a typical membrane potential recording before, during, and after exposing the myocyte to 5 µM KN-93. In the absence of the compound, a stable RMP of 44 mV was recorded. Application of KN-93 induced a 13-mV depolarization to 31 mV. As for the effects of KN-93 on K_V, RMP only partially recovered after washout of the drug (39 mV). Similar effects were consistently observed in a total of six experiments, and mean data are shown in Fig. 9B.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Effects of KN-93 on the resting membrane potential recorded in current clamp mode using the perforated patch technique. A, typical experiment showing the effects of application and washout 5 µM KN-93 (indicated by the thick bar) on the resting membrane potential recorded in a whole-cell current clamped portal vein myocyte with the nystatin technique. B, bar graph displaying mean data of RMP derived from six experiments similar to that shown in A. Each data point is a mean ± S.E.M. **, significantly different from each other with p < .01; *, significantly different from each other with p < .05 (one-way ANOVA test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our study reports for the first time that KN-93, a specific inhibitor of CamKII, is a potent blocker of voltage-dependent K⁺ current in vascular smooth muscle cells. The effects of KN-93, as those of the other CamKII inhibitor KN-62, appeared to be independent of changes in [Ca²⁺]_i and CamKII activity. These compounds were more potent at inhibiting late versus peak current during step depolarizations and they all enhanced the rate of inactivation of K_V. Finally, current clamp experiments revealed that 5 µM KN-93 reversibly depolarized portal vein smooth muscle cells by 15 mV, a result consistent with a KN-93-induced block of K_V.

KN-93 Primarily Inhibits Voltage-Dependent K⁺ Channels. Four distinct K⁺ currents have been described in freshly isolated rabbit portal vein smooth muscle cells: 1) an iberiotoxin- or TEA-sensitive Ca²⁺-dependent K⁺ current (K_Ca) that is carried by large conductance K⁺ channels (200 pS in symmetrical K⁺ gradient; Beech and Bolton, 1989a; Hume and Leblanc, 1989; Miller et al., 1993; Morales et al., 1996); 2) a fast transient voltage-dependent outward K⁺ current (I_to) (Beech and Bolton, 1989b); 3) a 4-AP-sensitive voltage-activated delayed rectifier K⁺ current (K_V); and 4) a glibenclamide-sensitive voltage-independent ATP- and GDP-sensitive K⁺ current (K_ATP) (Kajioka et al., 1991).

Potential Mechanisms of Block of K_V by KN-93. Ca²⁺-calmodulin and CamKII have been reported to regulate ion channel activity in several tissues. Activation of CamKII during repetitive stimulations that elevate [Ca²⁺]_i is responsible for the positive feedback control of Ca²⁺-induced enhancement of Ca²⁺ channels in rabbit cardiac (Anderson et al., 1994), toad stomach (McCarron et al., 1992), and rabbit portal vein (this study) smooth muscle cells and inactivation of Ca²⁺-dependent Cl channels in equine tracheal smooth muscle cells (Wang and Kotlikoff, 1997). Certain types of Ca²⁺-dependent K⁺ channels have been reported to be activated by Ca²⁺-calmodulin in renal medulla (Klaerke et al., 1987) and neurones (Onozuka et al., 1987). In contrast, other studies indicated that the inhibition of K⁺ channels mediated by calmodulin antagonists (McCann and Welsh, 1987; Kihira et al., 1990) may be caused by a direct interaction with the channels, thus ruling out a role for Ca²⁺-calmodulin in the gating process. In our study, KN-93 dose-dependently blocked K_V current with a potency (IC₅₀ = 270 nM for inhibition of late current) that was similar to its inhibitory action on rat brain CamKII (K_i = 370 nM; Sumi et al., 1991), and this effect was shown to be independent of [Ca²⁺]_i because cell dialysis with 10 mM EGTA could not prevent the inhibition. Although we cannot rule out the possibility that EGTA may have been unable to effectively lower subsarcolemmal Ca²⁺ levels or that a Ca²⁺-independent form of regulation of K_V by CamKII might be involved (Sumi et al., 1991; Singer et al., 1996), a role for CamKII seems unlikely because KN-92, an inactive substitute of KN-93 often used as a negative control, induced similar effects on K_V. Moreover, and in contrast to a preliminary study in murine colonic smooth muscle cells (Koh et al., 1999), prolonged cell dialysis with a specific peptide inhibitor of CamKII did not influence the magnitude and kinetics of K_V and failed to prevent the KN-93-induced inhibitory action on this current.