Abstract
The slowly activating delayed rectifier K+ current, IKs, is an important modulator of cardiac action potential repolarization. Here, we describe a novel benzodiazepine, [L-364,373 [(3-R)-1,3-dihydro-5-(2-fluorophenyl)-3-(1H-indol-3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one] (R-L3), that activates IKs and shortens action potentials in guinea pig cardiac myocytes. These effects were additive to isoproterenol, indicating that channel activation by R-L3 was independent of β-adrenergic receptor stimulation. The increase of IKs by R-L3 was stereospecific; theS-enantiomer, S-L3, blocked IKs at all concentrations examined. The increase in IKs by R-L3 was greatest at voltages near the threshold for normal channel activation, caused by a shift in the voltage dependence of IKsactivation. R-L3 slowed the rate of IKs deactivation and shifted the half-point of the isochronal (7.5 sec) activation curve for IKs by −16 mV at 0.1 μm and −24 mV at 1 μm. R-L3 had similar effects on cloned KvLQT1 channels expressed in Xenopus laevis oocytes but did not affect channels formed by coassembly of KvLQT1 and hminK subunits. These findings indicate that the association of minK with KvLQT1 interferes with the binding of R-L3 or prevents its action once bound to KvLQT1 subunits.
Repolarization from the plateau phase of the AP in ventricular myocytes is controlled by a delicate balance between inward and outward currents in the setting of a high membrane resistance. Important outward currents that determine repolarization are IKr and IKs (Sanguinetti and Jurkiewicz, 1990). Several class III antiarrhythmic agents block IKrand thereby prolong APD and the QT interval on the electrocardiogram. Excessive APD prolongation by these drugs causes LQT, which is associated with torsades de pointes, a ventricular tachyarrhythmia that can degenerate into ventricular fibrillation and cause sudden death.
LQT can also be inherited. The finding that mutations inHERG, the gene that encodes IKrchannels, cause inherited LQT (Curran et al., 1995;Sanguinetti et al., 1995, 1996a) provided a mechanistic link between acquired LQT and one form of inherited LQT. The most common form of LQT is caused by mutations in KvLQT1, a novel K+ channel gene (Wang et al., 1996). Expression of KvLQT1 in either Xenopus laevisoocytes or mammalian cell lines induced a K+current with biophysical properties unlike any known cardiac K+ current. Coexpression of KvLQT1with minK induced a current that was essentially identical to cardiac IKs, indicating that KvLQT1 and minK proteins coassemble to form IKs channels (Barhanin et al., 1996; Sanguinetti et al., 1996b). Thus, dysfunction of either IKr or IKs can increase the risk of cardiac arrhythmia and sudden death.
An activator of IKr or IKschannels might be useful for the treatment of LQT that results from excessive pharmacological block of these channels or from mutations in the genes that encode the channel proteins. We previously described the properties of L-735,821, a benzodiazepine that is a potent and selective stereospecific blocker of cardiac IKs (Salata et al., 1996). In this study and one preliminary report (Salata et al., 1997), we describe another 1,4 benzodiazepine, R-L3 (L-364,373), that is a stereospecific activator of cardiac IKs.
Experimental Procedures
Isolation of guinea pig ventricular myocytes.
Guinea pig ventricular myocytes were isolated as described previously (Salataet al., 1995). After isolation, the cells were stored in HBS containing 132 mm NaCl, 4 mm KCl, 1.8 mm CaCl2, 1.2 mmMgCl2, 10 mm HEPES, and 10 mm glucose, pH 7.2, at 24–26° until studied, usually within 8 hr after isolation.
Action potential studies.
Transmembrane potentials were recorded using conventional microelectrodes filled with 3 mKCl (tip resistances, 30–50 MΩ) using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) as described previously (Fermini et al., 1995). Cells were superfused with HBS maintained at 37° at a rate of 2 ml/min APs were evoked with brief current pulses (1 msec, 1.2 times threshold) delivered at a frequency of 1 Hz through the recording electrode using an active bridge circuit. Only cells showing normal AP configurations and resting membrane potentials equal to or more negative than −85 mV were used in this study. Cells were studied after a ≥5-min control period and after ≥5 min of superfusion with each concentration of drug. After reaching a steady state effect under each condition, 20 individual APs were sampled and digitally averaged. The APA50ms and APD90 were measured from the digitally averaged records. Concentration-response relationships were determined by measuring APs or currents in each cell under control conditions and during superfusion with successively increasing concentrations of a given drug.
Voltage-clamp of guinea pig ventricular myocytes.
Whole-cell voltage-clamp studies were performed using a List EPC-7 (Medical Systems, Greenvale, NY) or Axopatch 200A (Axon Instruments) amplifier as described previously (Fermini et al., 1995). Pipettes were made from square bore (1.0 mm outer diameter) borosilicate capillary tubing (Glass Company of America, Bargaintown, NJ). Pipettes were filled with 0.5 m K+ gluconate, 25 mm KCl, and 5 mm K2ATP to minimize “rundown” (Giles and Shibata, 1985) and had resistances of 3–7 MΩ (average, 5.5 ± 0.3 MΩ). Series resistance was compensated 40–70%. Currents were low-pass filtered (−3 dB at 0.2 kHz) before digitization at 1 kHz. IKs was measured during superfusion of the cells at a rate of 2–3 ml/min with normal HBS (35°) containing 0.4–1 μm nisoldipine to block L-type Ca2+ current and standard selective IKr blockers in 100-fold excess of the IC50 (e.g., 3μm MK-499) to completely block IKr (Sanguinetti and Salata, 1996). Cells were voltage clamped at a Vh of −50 mV to inactivate INa. Time-dependent IKsamplitude was measured as the difference from the initial instantaneous current, after the settling of the capacity transient, to the final current level at the end of a depolarizing pulse. IKtail was measured as the difference from the holding current level to the peak tail current amplitude on return to Vh. IKtail was normalized to the maximum measured amplitude (IKtail-max) after 7.5-sec pulses. Averaged data were fit to a Boltzmann distribution of the form: IKtail/IKtail-max = 1/(1 + exp[(V1/2 − Vt)/k]) with a nonlinear least-squares fitting routine (Origin; Microcal Software, Northampton, MA) to estimate the half-point (V1/2) and slope factor (k) for this relationship. The time courses of IKs activation and deactivation were fit with a double exponential relationship of the form: It = A0 + A1 e−t /τ1 + A2 e−t /τ2 using a Chebeshev noniterative fitting technique (pCLAMP; Axon Instruments).
ICa was recorded using external and internal (pipette) solutions designed to minimize currents through Na+ and K+ channels and reduce rundown (Xu and Lee, 1994). Cells were superfused with a solution containing 157 mm TEACl, 5 mmCaCl2, 0.5 mmMgCl2, and 10 mm HEPES, pH adjusted to 7.4 with CsOH (24–26°). Microelectrodes were fashioned from borosilicate capillary tubing (1.5 mm absorbance and filled with the following solution: 151 mm CsOH, 10 mm l-aspartic acid, 20 mm taurine, 20 mm TEACl, 10 mm EGTA, and 5 mmglucose, pH adjusted to 7.5 with H3PO4; 0.4 mmNa2GTP and 5 mm MgATP were added just before use. The filled pipettes had resistances ranging from 2 to 4 MΩ. Series resistance was compensated by 80%. Because we did not observe bimodal I-V relations in our experiments, T-type Ca2+ channels most likely contributed negligible current; therefore, the ICa measured in the current study was considered to be L-type Ca2+ current.
Culture and voltage clamp of AT-1 cells.
The original mouse atrial tumor (AT-1) mouse colonies were established by Dr. Loren Field (Krannert Institute of Cardiology, Indianapolis, IN) (Field, 1988), and the lineage at Merck Research Laboratories was established from tumor cells that were provided by Dr. Dan Roden (Vanderbilt University, Nashville, TN). AT-1 cells were propagated in vivo, and their isolation and culturing were conducted as described previously (Delcarpio et al., 1991; Yang et al., 1994;Jurkiewicz et al., 1996).
For voltage-clamp studies, AT-1 cells were trypsinized to remove them from the culture dishes and stored in PC-1 culture medium (22–24°). Outward K+ currents were recorded in normal HBS at 22–24° using standard whole-cell voltage-clamp techniques within 14 hr of isolation. Pipettes were filled with a solution containing 110 mm KCl, 5 mm K-BAPTA, 5 mmK2ATP, 1 mmMgCl2, and 10 mm HEPES, pH 7.2, and had resistances of 3–7 MΩ (average, 5.5 ± 0.3 MΩ). All cells were round in appearance, had large outward tail currents and resting membrane potentials (RMP) negative to −35 mV, and did not beat spontaneously. INa and T-type calcium currents were inactivated by voltage-clamping the cells to a Vh of −40 mV. ICa was blocked with 0.4 μm nisoldipine.
cRNA injection and voltage-clamp of oocytes.
The isolation and maintenance of X. laevis oocytes, in vitrotranscription of KvLQT1 and hminK cRNA, and its injection into oocytes were performed as described previously (Sanguinetti et al., 1995, 1996b). Stage V and VI oocytes were injected with 11.5 ng of KvLQT1 cRNA (46 nl of a 250 ng/μl solution) alone or coinjected with 11.5 ng of KvLQT1plus 1.25 or 0.1 ng of hminK cRNA. Currents were recorded 2–4 days later using standard two-microelectrode voltage-clamp techniques and a Dagan TEV-200 amplifier. Oocytes were bathed at room temperature (22–25°) in a solution containing 94 mmNaCl, 2 mm KCl, 2 mmMgCl2, 0.1 mmCaCl2, and 5 mm HEPES, pH 7.6.
Materials.
R-L3 (Fig. 1) was prepared as described previously by Evans et al. (1987). Its enantiomer, S-L3, was prepared by the same procedure described for R-L3, with l-tryptophan acid chloride hydrochloride used in place of the d-isomer: 1H NMR (CDCl3) identical to that of R-L3. The chemical and chiral purity of R-L3 and S-L3 were determined to be >99%. S-L3: high performance liquid chromatography (Vydac C-18, 15 × 0.46 cm, 16 min gradient 95:5 to 5:95 0.1% H3PO4/H2O:CH3CN, 1.5 ml/min, 215 and 254 nm) retention time (rt) = 11.0 min, >96%, coelutes with R-L3. high performance liquid chromatography (Chiralcel absorbance 25 × 0.46 cm, 90/10 hexane/EtOH, 1.5 ml/min, 280 nm) rt = 9.95 min, 98.2%, contains <1% of R-L3 (R-L3: rt = 10.94 min, 99.6%, contains <0.5% of S-L3). TLC (silica, 10% Et2O in CH2Cl2): single component, Rf = 0.43, coelutes with R-L3. Calc. for C25H20FN3O: C 75.55, H 5.07, N 10.57; found: C 75.57, H 5.17, N 10.46.
Compounds were dissolved in dimethylsulfoxide at a stock concentration of 1 or 10 mm and diluted directly into test solutions. Serial dilutions were used to achieve the final test concentrations. Dimethylsulfoxide at the concentrations used had no significant effect on any of the parameters measured in these studies. Nisoldipine (a gift from Miles Pharmaceuticals, New Haven, CT) was prepared as a 4 mm stock solution in dimethylsulfoxide and diluted as needed.
Statistics.
Data are expressed as mean ± standard error. Concentration-dependent changes in AP parameters and individual ionic currents were assessed by repeated-measures analysis of variance.Post hoc comparison of the treatment with the control mean values were made with Dunnett’s t test to determine significant changes between the control and test group mean values. Statistical comparisons for the time constants of IKs activation and deactivation were made using a paired t test. A one-tailed probability (p < 0.05) was considered significant.
Results
R-L3 decreases APD of cardiac myocytes.
R-L3 (0.1–1.0 μm) caused a concentration-dependent shortening of APD. Fig. 2A shows a representative example of APs recorded at a stimulus frequency of 1 Hz. R-L3 significantly decreased APD50 and APD90without significantly affecting other AP parameters (Table1). Shortening of APD was maximal at 1 μm; APD90 was decreased at concentrations of 1 and 10 μm R-L3 by 14.2 ± 1.6% and 13.8 ± 4.0%, respectively.
Stimulation of β-adrenergic receptors can also shorten APD of cardiac myocytes (Carmeliet and Vereecke, 1969; Sanguinetti et al., 1991). Therefore, we determined whether the decrease in APD by R-L3 was mediated through the same or parallel pathway. At a concentration of 30 nm, Iso decreased APD90 by 12.9 ± 2.9%. Iso also increased APA50ms by 6.6 ± 2.9% (Fig. 2B), presumably by enhancement of L-type Ca2+ current (Kass and Wiegers, 1982). The addition of 1 μm R-L3 in the presence of 30 nm Iso decreased APD90 further (26.9 ± 1.4%) and diminished the increase in APA50ms. Block of β-adrenergic receptors with 100 nm timolol did not alter configuration (Fig. 2C) but prevented the effects of 30 nm Iso (data not shown). In the continued presence of timolol, the addition of 1 μm R-L3 decreased APD90 by 14.6 ± 2.2%, very similar to the effect observed as in the absence of timolol. Thus, the decrease in APD by R-L3 is additive to the effect mediated by β-adrenergic stimulation.
R-L3 increases IKs of guinea pig myocytes in a concentration-dependent and stereospecific manner.
R-L3 is structurally related to L-735,821, a benzodiazepine that selectively blocks IKs and prolongs APD of guinea pig ventricular myocytes (Salata et al., 1996). Therefore, we reasoned that R-L3 might shorten APD of guinea pig myocytes by activating IKs.
The effect of R-L3 on IKs was measured under voltage-clamp conditions using 3-sec depolarizations to a test potential (Vt) of −10 mV from a Vh of −50 mV (Fig.3A). In contrast to the previously reported effect of L-735,821, R-L3 increased IKs. R-L3 enhanced IKsmeasured at −10 mV at concentrations as low as 30 nm and had a maximal effect at 1 μm. At this concentration, IKs was increased by a factor of 17 ± 5 (six cells). At a concentration of 3 or 10 μm, the percentage increase in IKs by R-L3 was less than that observed for 1 μm (Fig. 3B). This diminished response at high concentrations was caused by a time- and voltage-dependent block of IKs that was most obvious during long pulses. For example, IKs was increased by 10 μm R-L3 during the first few seconds of a 7.5-sec pulse to +50 mV. However, when the depolarization exceeded ∼3 sec, the current measured in the presence of R-L3 was reduced compared with control (Fig. 3C). R-L3 increased time-dependent IKs at the end of 7.5-sec pulses to potentials <+10 mV but decreased IKs at more positive potentials (Fig. 3D). Thus, the biphasic concentration-response relationship for the effects of R-L3 on IKs for 3-sec pulses to −10 mV (Fig. 3B) reflects the dual effects of the drug: activation that predominates at low concentrations and voltage-dependent block at higher concentrations.
The increase of IKs by R-L3 was stereospecific. S-L3 blocked IKs at all concentrations (1–10 μm) and test potentials (−10 to +50 mV) examined. At concentrations of 1 and 10 μm, S-L3 blocked IKs measured at the end of a 1-sec test pulse to +50 mV by an average of 14.8 ± 4.3% and 68.8 ± 3.4% (five cells).
R-L3 shifts the voltage dependence of activation and slows deactivation of IKs.
The voltage dependence of IKs activation was estimated using 7.5-sec depolarizing steps from a Vh of −50 mV (Fig.4A). The amplitude of the tail currents was normalized relative to the maximum amplitude and fit to a Boltzmann function (Fig. 4B). Because IKs does not achieve a steady state, even during extremely long pulses (Hice et al., 1994), the activation curves are isochronal. In control, the V1/2 was 19.2 ± 1.6 mV, and kfor this relationship was 11.0 ± 1.2 mV (five cells). In these same cells, R-L3 shifted V1/2 to 3.0 ± 0.8 mV at 0.1 μm and −4.9 ± 3.4 mV at 1 μm but had no effect on k. The maximally activated IKs measured at a Vt of +60 mV (896 ± 196 versus 953 ± 183 pA, 1 μm) was slightly but not significantly increased by R-L3. Likewise, after pretreatment with 100 nm timolol, 1 μm R-L3 shifted the V1/2 by −19 mV without affectingk, indicating that its effect was independent of β-adrenergic stimulation. These results suggest that the primary mechanism of the increase in IKs by R-L3 is an effect on channel gating.
The onset of IKs activation, after a short delay, was best described by a two-exponential function. The fast time constants of activation were slightly, but not significantly, faster in the presence of 1 μm R-L3. This effect was likely due to the negative shift in the voltage dependence of channel activation. In contrast to the modest effect on the kinetics of activation, R-L3 greatly slowed the rate of IKs deactivation (Fig.4A). The kinetics of deactivation were determined at potentials of −60 to −10 mV after a 3-sec prepulse to +30 mV from a Vh of −50 mV. The deactivation of IKs was best described by a two-exponential function before and after the addition of R-L3. R-L3 significantly increased the fast (τfast) and the slow (τslow) time constants of deactivation (Fig.5). The slowing of the rate of deactivation by R-L3 represents an additional mechanism that would increase outward current during repolarization of a cardiac AP.
R-L3 activates IKs independent of β-adrenergic receptor activation.
IKs was activated by 0.5-sec pulses to a Vt ranging from −40 to +50 mV. Iso (10 nm) alone increased IKsby 1.75-fold. The addition of 1 μm R-L3 produced a further increase in IKs, slowed the rate of deactivation, and shifted the threshold for current activation to more negative potentials (Fig. 6). The effects of R-L3 persisted after washout of the Iso. Similar effects were observed when exposure of cells to R-L3 preceded the addition of Iso. Thus, similar to the decrease in APD caused by R-L3, the increase in IKs by R-L3 was additive to that caused by β-adrenergic receptor stimulation.
R-L3 activates cloned human KvLQT1 channels expressed in X. laevis oocytes.
At a concentration of 1 μm, R-L3 increased KvLQT1 elicited with 2-sec pulses to potentials ranging from −70 to +60 mV (Fig. 7, A and B). This increase can partially be accounted for by a −10-mV shift in the voltage dependence of activation caused by the drug (Fig. 7C). R-L3 also slowed the kinetics of KvLQT1, an effect that is easily observed when the time-dependent currents recorded before and after exposure to 1 μm R-L3 are superimposed and scaled to match peak current (Fig. 8A). Activation of KvLQT1 current is best described by a two-exponential function. The effect of R-L3 on these two components varied with Vt. R-L3 slowed the rate of the fast component at Vt ≥0 mV (Fig. 8B), but increased the rate of the slow component of activation at Vt ≥−30 mV(Fig. 8C). The net effect of R-L3 was to slow the rate of KvLQT1 activation because of a reduction in the relative amplitude of the fast component of activation over the entire voltage range that was examined (Fig. 8D). R-L3 also slowed the rate of KvLQT1 deactivation when assessed at voltages negative to −40 mV (Fig. 8E). Thus, R-L3 increased the magnitude of KvLQT1, shifted the voltage dependence of its activation, and slowed the rates of activation and deactivation. These effects of R-L3 on KvLQT1 current are similar to those observed for IKs recorded in guinea pig ventricular myocytes.
R-L3 activates cloned human IKs currents depending on hminK/KvLQT1 ratio.
IKs currents are formed by coassembly of KvLQT1 and minK subunits (Barhanin et al., 1996; Sanguinetti et al., 1996b). Therefore, in addition to its effects on KvLQT1 channels, we determined the effects of R-L3 on KvLQT1 plus hminK (IKs) currents expressed in X. laevis oocytes. When oocytes were injected with 11.5 ng of KvLQT1 and 1 ng of hminKcRNAs, amounts similar to previous studies, we expected to observe an increase in the magnitude of cloned IKs similar to that described above for IKs recorded from guinea pig myocytes. Surprisingly, we found that R-L3 had no obvious effect on channels formed by coassembly of KvLQT1 plus hminK (Fig.9). The only statistically significant effect of the drug was a slowing of the rate of deactivation. At −50 mV, deactivation was 470 ± 18 msec in control and 507 ± 12 msec after 1 μm R-L3 (p < 0.05). We observed a similar lack of effect of R-L3 on expressed IKs when either human or guinea pigminK cRNA alone was injected into X. laevisoocytes (data not shown), Presumably, these currents represent channels formed from coassembly of exogenous minK and endogenous KvLQT1. These data indicate that association of KvLQT1 with minK subunits prevents the activation of channel activity by R-L3 that occurs when only KvLQT1 channels are overexpressed in oocytes.
To test this hypothesis further, the ratio of hminK/KvLQT1 subunits was reduced by injecting oocytes with 11.5 ng of KvLQT1 and 0.1 ng of hminK cRNA. Under these conditions (Fig.10), the induced current activated at a rate faster than IKs but slower than KvLQT1 alone (compare with Figs. 7 and 9), suggesting that not all channels were heteromultimeric. In this case, R-L3 caused an increase in current in all cells (seven cells). On average, R-L3 increased peak outward current by 28% at +40 mV.
R-L3 blocks IKr and ICa but not IK1.
To determine the selectivity of R-L3, we measured its effects on three other currents, IKr, IK1, and ICa, that modulate cardiac APD.
IK1 and ICa were measured in guinea pig isolated ventricular myocytes. R-L3 at 10 μm had no significant effect on IK1. For example, IK1 at −60 mV was 5.5 ± 1.1 pA/pF in control and 5.7 ± 1.2 pA/pF after the addition of R-L3 (five cells). R-L3 had no significant effect on ICa at 1 μm, but at 10 μm it reduced peak ICa at +20 mV by 43.6 ± 6.6% (nine cells; Fig.11). The block of ICa was not use-dependent. In two cells, block of ICa by 10 μm R-L3 during trains of 30 pulses applied to +20 mV at rates of 1 and 3 Hz initially was 57% and 61%, respectively, and was unchanged (59% and 60%) at the end of the pulse trains.
Because R-L3 caused a negative shift in the voltage-dependence of IKs activation, IKr could not satisfactorily be measured in isolation from IKs in guinea pig myocytes. Therefore, the effects of R-L3 on IKr was determined in mouse AT-1 myocytes (Fig. 12). These cells have a large IKr but no measurable IKs (Yang et al., 1994). R-L3 blocked IKr tail currents after a 1-sec test pulse to +20 mV by 21 ± 6% and 53 ± 3% (three cells) at concentrations of 1 and 10 μm, respectively.
Discussion
At concentrations of ≤1 μm, R-L3 shortened APD of cardiac myocytes by selective activation of IKs. At membrane potentials and pulse durations typical for a cardiac AP, the most important mechanisms of action of R-L3 were a negative shift in the voltage dependence of activation and a slowing of IKs deactivation. R-L3 also caused a modest increase in IKs beyond what could be explained by these two mechanisms. The molecular mechanism of these effects on IKs current-gating is not known, but it is not mediated through the β-adrenergic receptor activation pathway.
At concentrations of >1 μm, the effect of R-L3 on APD would reflect multiple mechanisms, including activation of IKs (especially at potentials of <0 mV), block of IKr, and block of L-type ICa. Block of IKr would lengthen APD, whereas block of ICa would contribute to a shortening of APD. Because APD90was unchanged, whereas APD50 was further shortened and the plateau height (APA50ms) was reduced when R-L3 was increased from 1 to 10 μm, it is likely that the block of ICa was more important than the block of IKr at 10 μm in guinea pig ventricular myocytes. A high concentration (10 μm) of R-L3 also caused a block of IKs after long pulses to very positive potentials. However, even at 10 μm, R-L3 would be expected to cause only an increase in IKs during the limited time of depolarization of a cardiac AP. We could not test the effects of R-L3 at concentrations of >10 μm because of its limited aqueous solubility.
We compared and contrasted the effects of R-L3 and Iso on configuration. These studies revealed a similar shortening of APD90 but other important differences, especially on plateau height. Effects of β-adrenergic receptor stimulation on APs are difficult to interpret because of the multitude of effects on ion channels and pumps. Nevertheless, the persistent effects of R-L3 in the presence of timolol, a β-adrenergic receptor blocker, demonstrate that its effects are not mediated via this signaling pathway.
The findings that R-L3 activated IKs at low concentrations but blocked IKs at high concentrations, as did the S-enantiomer, suggest that there may be multiple binding sites for L-3 on the IKs channel. Multiple binding sites have been proposed to explain the dual action of dihydropyridines such as Bay K 8644 on the L-type Ca2+ channel (Brown et al., 1986; Kokubun et al., 1986).
R-L3 increased the magnitude of cloned KvLQT1 current, but its activation of human IKs channels formed by coassembly of KvLQT1 and hminK subunits depended on their ratio. When minK and KvLQT1 were coexpressed at relatively high ratio, such that the expression of minK was not limiting, R-L3 had no significant effect on IKs. However, when coexpressed at a 10-fold lower ratio, where minK was limiting, R-L3 increased IKs. The resulting currents at the low minK/KvLQT1 ratio were larger and activated at a rate slower than that of the current induced by KvLQT1 alone but were faster than those induced by the high subunit ratio. The stoichiometry of IKs channels is unknown. Because KvLQT1 alone or coexpressed with minK can form functional channels, it may be possible for minK subunits to coassemble with individual KvLQT1 subunits or KvLQT1 tetramers in variable ratios and thereby impart differing biophysical characteristics and pharmacological sensitivity. Alternatively, there may be only one functional IKs channel type with a fixed stoichiometry of KvLQT1 and minK subunits, and these may coexist in variable ratios with KvLQT1 homotetramers. A clearer understanding of this stoichiometry may help to explain why the effects of the drug on guinea pig IKs are better mimicked by cloned KvLQT1 channels, whereas the biophysical properties of IKs are more closely mimicked by channels formed by coassembly of KvLQT1 + hminK. Nevertheless, these findings indicate that the binding site for R-L3 is located on the KvLQT1 subunit and that coassembly with hminK diminishes or abolishes the agonist activity of R-L3. These studies are consistent with, but not proof of, the ideas that minK and R-L3 bind to a common region of KvLQT1 subunits and that association of minK with KvLQT1 precludes the drug-induced alteration of gating kinetics observed when only KvLQT1 channels are overexpressed in oocytes. Regardless of the exact mechanism, these findings suggest the possibility that IKsrecorded in cardiac myocytes represents the sum of current mediated by KvLQT1 homotetrameric channels and heteromultimeric channels formed by coassembly of KvLQT1 plus minK subunits.
This and all studies of IKs face the same possible limitations, including the potential for K+ accumulation during long depolarizing pulses (Boyett et al., 1980, but see also Sanguinetti and Jurkiewicz, 1990), a lack of steady state activation of IKs, and interference or overlap with other cardiac currents. Our voltage-clamp protocols and conditions were designed to reduce or eliminate these concerns. To measure activation of IKs channels, relatively long pulse durations of 7.5 sec were used as in previous studies (Sanguinetti and Jurkiewicz, 1990) to approach steady state activation, whereas longer pulses were avoided to minimize the potential for K+ accumulation. IKrblockers were used in excess (100 × IC50 values at 4 mm [K+]o) to completely block IKr. Although an increase in [K+]o has been reported to decrease the potency of IKr blockers (e.g., dofetilide; (Yang and Roden, 1996), the 100-fold excess used to block IKr in this study would not be overcome by potential elevations of [K+]o. Most currents other than IKs were also eliminated by pharmacological blockade or inactivation (or both) with Vh.
A drug that activates IKs could be beneficial for the treatment of certain arrhythmias. The proarrhythmic potential of class III antiarrhythmic drugs that block IKrprompted the search for agents that would produce more modest prolongation of APD at high doses. The proarrhythmic liability associated with block of IKr currents was recently confirmed by the finding that mutations in HERG, the gene encoding IKr channel subunits, can cause inherited LQT (Curran et al., 1995). Recently, IKs blockers have been described that lengthen APD and have antiarrhythmic effects in animal models. Chromanol 293B (Busch et al., 1996) and the more potentL-735,821, a 1,4-benzodiazepine (Salata et al., 1996), inhibit IKs and cause a self-limiting prolongation of APD that is less than that produced by IKr blockers. However, mutations in KvLQT1 can also cause inherited LQT (Wang et al., 1996; Neyroudet al., 1997). This latter finding could dampen enthusiasm for the development of IKs blockers for prophylactic treatment of ventricular tachyarrhythmias. If IKr or IKs blockers are used as antiarrhythmic drugs, it would be useful to have a channel activator that could reverse the effects of overdose. Moreover, pharmacological activation of IKs might also be useful for treatment of inherited LQT and abnormally delayed repolarization associated with heart failure (Beuckelmann et al., 1993; Tomaselli et al., 1994).
In summary, we described the properties of R-L3, a novel benzodiazepine that selectively activates IKs in myocytes at low concentrations. This compound represents a new pharmacological probe for the study of IKs in cardiac myocytes. In addition, R-L3 can be used as a probe to define the physiological role of IKs and KvLQT1 currents in other tissues, such as the pancreas (Wang et al., 1996) and stria vascularis of the inner ear (Neyroud et al., 1997).
Acknowledgments
We thank Mr. Carl Homnick for conducting the chiral high performance liquid chromatography studies and Qing Xu for preparing the cRNA.
Footnotes
- Received July 10, 1997.
- Accepted January 29, 1998.
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Send reprint requests to: Dr. Joseph J. Salata, Department of Pharmacology, Merck Research Laboratories, Sumneytown Pike, P.O. Box 4, WP46–300, West Point, PA 19486. E-mail:joseph—salata{at}merck.com
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This study was supported in part by U.S. Public Health Service Grant RO1-HL55236 (M.C.S.).
Abbreviations
- AP
- action potential
- APA50ms
- action potential amplitude measured at 50 msec after the upstroke
- APD90
- action potential duration at 90% repolarization
- APD50
- action potential duration at 50% repolarization
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- HBS
- HEPES-buffered saline
- Ica
- L-type Ca2+ current
- IK
- delayed rectifier K+ current
- Ikr
- rapidly activating component of delayed rectifier K+ current
- IKs slowly activating component of delayed rectifier K+ current
- IK1, inward rectifier K+ current
- Iktail
- IKtail current
- Iktail-max
- maximum amplitude IKtail current
- Iso
- isoproterenol
- I-V
- current-voltage
- [K+]o
- extracellular K+concentration
- LQT
- long QT syndrome
- R-L3
- (3-R)-1,3-dihydro-5-(2-fluorophenyl)-3-(1H-indol-3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one
- Vh
- holding potential
- Vt
- test potential
- The American Society for Pharmacology and Experimental Therapeutics