Halothane Inhibition of Recombinant Cardiac L-type Ca²⁺ Channels Expressed in HEK-293 Cells

HEK-293 cells were transiently cotransfected by calcium phosphate precipitation with complementary DNAs encoding α^1C tagged with green fluorescent protein on the N termini to serve as a reporter, and rabbit heart β^2a, with and without rabbit skeletal muscle α²/δ¹ subunits. Cells were initially cotransfected with α^1C, but this method yielded low transfection efficiencies (< 5%) leading to poor experimental efficiency. To facilitate the identification of cells expressing α^1C, transfections were conducted with α^1C tagged with green fluorescent protein. Cells expressing the fusion protein were identified using fluorescence microscopy. Transfection efficiencies remained low (< 5%), but experimental efficiency was markedly enhanced.

Whole cell barium currents were recorded at room temperature 2–3 days after transfection from individual cells expressing α^1C tagged with green fluorescent protein as identified by fluorescence microscopy. This study focused on halothane effects on voltage-dependent inactivation, and therefore, Ba²⁺ was used as the charge carrier because it weakly triggers calcium-dependent inactivation. Cells were voltage clamped using the whole cell configuration of the patch clamp technique at room temperature. An Axopatch 200A amplifier (Axon Instruments, Union City, CA) was connected to an IBM-compatible computer running the Axobasic (Axon Instruments) environment and software of our own design. Bath solution contained 130 mm N -methyl-d-glucamine-aspartate, 1 mm MgCl², 10 mm glucose, 10 mm 4-aminopyridine, 10 mm HEPES, and 20 mm BaCl², pH 7.3–7.4 with 1 m N -methyl-d-glucamine-aspartate. Internal solution contained 140 mm N -methyl-d-glucamine-methanesulfonate, 5 mm EGTA, 1 mm MgCl², 4 mm Mg-adenosine triphosphate, and 5 mm HEPES, pH 7.2–7.3 with CsOH. Currents were sampled at 10 kHz and filtered at 2 kHz. Currents were measured (−70 mV to −80 mV holding potential) in the presence of halothane (0–5 mm), and leak and capacitive transients were subtracted by a P/8 protocol. Interstimulus intervals were greater than 15 s.

The cell being investigated was continuously superfused with solutions delivered by a local solution changer, which involved a closed system constructed entirely of stainless steel, glass, polytetrafluoroethylene tubing, and glass syringes with a syringe pump. Currents were obtained in control, drug, control sequence, and data were included provided the bracketing peak control current was greater than 90% of initial control. Using the internal and external solutions as described, pipette resistances were 2–5 MΩ, which was compensated by greater than 60%. Time constant for charging membrane capacitance was approximately 0.6 ms. The VA agent halothane in a thymol-free preparation (Halocarbon, River Edge, NJ) was diluted from a saturated solution (17.5 mm) and dissolved in the bath solution before being loaded into the local solution changer.

Curve fitting and statistical analysis was performed with ORIGIN software (OriginLab, Northampton, MA). The data are shown as mean ± SEM, unless otherwise stated.

The maximum channel conductance (G^peak) was calculated using

where V is the voltage of the activating depolarization, I^peak is the current maximum during the activating depolarization, and V^rev is the reversal potential as estimated by extrapolating the quasi-linear portion of the current–voltage (I-V) relation near zero current at positive voltages.

Voltage dependence of activation was quantified by first relating peak G^peak normalized by maximum G^peak to the activating voltage, which was followed by a quantitative description of this relation using a two-state Boltzmann equation of the form

where V is voltage, V^1/2 is the midpoint voltage, and dx is the slope factor.

To quantify the kinetics of activation and inactivation, both time courses were fit simultaneously with

where I(t) is the current magnitude at time t, and A, B, and C are coefficients of the exponential components with time constants τ^a, τ^b, and τ^c, respectively.

Concentration–inhibition relations were fit with a sigmoidal equation of the form

where y is the response variable, [A] is the halothane concentration, IC⁵⁰ is the concentration at 50% inhibition from control, and n is the slope factor related to the Hill coefficient.

Transmembrane charge transfer (Q^T) was calculated by integrating the area under the current response from the time it crossed the baseline after the capacity transient to the end of the triggering depolarization (+10 mV, 300 ms). The relative contribution of I^peak changes were approximated by calculating fractional I^peak reduction relative to control. This approach determines Q^T occurring in the absence of alterations in current time course caused by changes in activation and inactivation kinetics. Reductions in Q^T that were unaccounted for by changes in I^peak were attributed to changes in gating kinetics reflected in alterations in current time course. Relative contributions of I^peak and kinetics were then calculated straightforwardly, assuming these two represented all contributing factors.

shows a representative family of current responses triggered by a range of voltage steps from single HEK-293 cells transfected with either α^1Cβ^2a or α^1Cβ^2aα²/δ¹ subunits. The peak I-V relations () show that maximum I^peak was evoked at approximately +20 mV for α^1Cβ2a channels, whereas that for α^1Cβ^2aα²/δ¹ channels is leftward shifted by approximately 10 mV, consistent with previous reports. Coexpression of α²/δ¹ with α^1Cβ^2a subunits enhanced inactivation as reported by the current at the end of a single 300-ms depolarization (I³⁰⁰; , inset) in single cells depolarized to +30 mV as well as in grouped data over a range of voltages (). These results indicate that voltage-dependent inactivation is potentiated by the presence of the α²/δ¹ subunit in accord with previous reports16,,21 and that transfection resulted in the functional expression of LCCs with the expected subunit composition.

We next investigated the concentration dependence of halothane depression of I^peak and I³⁰⁰. Currents triggered by +10-mV depolarizations were studied because this depolarization is near the maximum of both I-V relations and both measures show little voltage dependence between +10 and +30 mV (see Results, Halothane Effects on Current– and Conductance–Voltage Relations). Halothane (1 mm) reversibly depressed I^peak and altered the time course of currents elicited from individual cells expressing α^1Cβ^2a and α^1Cβ^2aα²/δ¹ channels (, top). The magnitude of I^peak depression was confirmed in grouped data and over a range of halothane concentrations (, left). Responses normalized to I^peak show that inactivation is enhanced by halothane for both channels, although it seemed greater with coexpression of α²/δ¹ (, bottom). The magnitude of I³⁰⁰ is influenced by changes in I^peak, and therefore, I³⁰⁰ was plotted after normalization for I^peak (I³⁰⁰/I^peak; , right). These relations show that effects on inactivation of α^1Cβ^2aα²/δ¹ are markedly greater than α^1Cβ^2a as shown by more than 10-fold difference in IC⁵⁰ (1.38 and 14.5 mm, respectively). The results confirm halothane enhancement of inactivation for both channels and potentiation of this effect in the presence of α²/δ subunit. Both I^peak and I³⁰⁰ relations show greater halothane sensitivity in the presence of α²/δ¹. This suggests that α²/δ¹ not only enhances control inactivation but also potentiates halothane inhibition of I^peak. Overall, the results suggest that phenotypic changes associated with α²/δ¹ coexpression potentiate halothane effects on I^peak and apparent inactivation and are consistent with the view that voltage-dependent inactivation is a target of VA action.

Reductions in cellular Ca²⁺ entry representing transmembrane charge transfer produced by VA inhibition of LCCs leads to depressed cardiac contractility.1,,2,,3,,4 Transmembrane charge transfer is determined by current magnitude (I^peak) and time course (gating kinetics), which are both affected by halothane (). We investigated the impact of changes in I^peak and kinetics on computed transmembrane charge transfer (Q^T; see Methods ) to gain insight into their relative contributions () to reductions in cardiac contractile state. shows representative current responses for both channels where the charge transferred (Q^T) during a triggering stimulus is proportional to the area under the current time course (hatched area). Halothane (1 mm) markedly reduced Q^T in both channels. Charge transferred (Q^T) was steeply dependent on halothane concentration for both channels (, left). Approximately 1½ minimum alveolar concentration (MAC) (approximately 0.6 mm) of halothane depressed Q^T by nearly 50% in α^1Cβ^2aα²/δ¹ channels, whereas a similar effect in α^1Cβ^2a channels required more than threefold greater halothane concentration (> 2 mm). Changes in I^peak accounted for more than 75% of the change in Q^T over the entire concentration range, whereas halothane effects on gating (acceleration of activation and inactivation) contributed less than 25% for both channels (, right). This is consistent with the crudely similar current time courses in control and halothane evident after normalization for I^peak (, bottom). The similarity of I^peak relations despite clear differences in inactivation kinetics between these channels strongly supports a primary role of I^peak in determining charge transfer. Q^T was computed over a period of constant membrane potential (+10 mV, 300 ms). Mean open channel probability is proportional to Q^T, because macroscopic current is the product of single channel open probability, channel number, and unitary current that is unaltered by halothane. Therefore, from this analysis, halothane depression of I^peak plays a primary role in the reduction of Q^T and mean open probability and likely plays an important role in LCC-mediated depression of cardiac contractility. Notably, halothane effects on channel gating (activation and inactivation) make minor contributions to reductions in Q^T and mean open probability. The results point to the importance of halothane interactions with channel states present at resting membrane potentials, which may underlie reductions in I^peak.

We next examined halothane effects on channel function over a range of triggering potentials. shows families of current responses triggered over a range of triggering potentials in control and 1 mm halothane for both channel types. Inspection of resultant I-V relations indicates that halothane depressed I^peak values without apparent changes in the nature of these relations (). This indicates that halothane reduced the number channels available to activate during a triggering depolarization. Halothane reduces time to peak (see Results, Halothane Alters Activation and Inactivation Kinetics), which may be explained by acceleration of activation; we therefore first investigated the voltage dependence of activation by constructing peak conductance (G^peak)–voltage relations. G^peak reports the peak channel open probability. G^peak was computed from peak ionic current measurements and the driving force as determined from the difference between test voltage and measured reversal potential (see Materials and Methods). For each channel type, after normalization for maximum G^peak, control and halothane relations show a similar dependence on voltage, indicating that the voltage dependence of peak open probability is unaltered by halothane (). To further investigate the effects of halothane on the number of activatable channels, we examined the voltage dependence of halothane depression of I^peak (). In channels with and without the α²/δ¹ subunit, halothane’s effect showed little voltage dependence, consistent with halothane’s primary effect of alteration in the number of activatable channels. Statistical means testing demonstrated no detectable difference in this endpoint between 10- and 20-mV triggering potentials for each channel type. The magnitude of apparent inactivation enhancement (300-ms depolarizations) produced by halothane was potentiated by increasing depolarization until reaching a seeming plateau at approximately 10 mV for both channels ().

Normalized current traces indicate that halothane reduced the time to I^peak for both channels (, bottom, insets). Time to I^peak, determined by inspection, was reduced by halothane for α^1Cβ^2a (control, 54.8 ± 6.9 ms; halothane, 37.0 ± 4.3 ms; P  < 0.01) and α^1Cβ^2aα²/δ¹ (control, 21.8 ± 3.7 ms; halothane, 14.1 ± 2.3 ms; P  < 0.01). G^peak reflects peak open channel probability, and time to peak of macroscopic current reports the time point of this event, presuming that unitary conductance remains constant. Both measures are primarily dependent on channel opening and activation and are opposed by channel closure, deactivation, and inactivation. As a result, it is notable that halothane reduced time to I^peak unaccompanied by changes in the form of the conductance–voltage relation. We therefore were interested in determining the effects of halothane on channel activation and inactivation through the quantitative analysis of the current time course. Current time courses were fitted with multiexponential functions, which provides for the separation and quantitation of activation and inactivation kinetics (see Materials and Methods). The α^1Cβ^2a current time course was well fit by a triexponential function (equation 3), where activation was described by two exponential components (time constants: fast [τ^f] and slow [τ^s]) and inactivation by single component (time constant: τⁱ) (). Both components of activation were accelerated by halothane () without changes in relative proportion (, bottom left). Halothane also accelerated inactivation (, bottom right). These effects were generally observed over a range of triggering voltages.

The time course of currents carried by α^1Cβ^2aα²/δ¹ were also well fit by a triexponential function (equation 3) but with monoexponential activation (time constant: τ^a) and biexponential inactivation (time constants: τ^s and τ^f) (), consistent with previous reports. Both activation and inactivation were accelerated by halothane (). We next investigated the effects of triggering voltages on halothane acceleration of activation and inactivation. Halothane acceleration of activation was present throughout the voltage range, although this affect appeared less at greater depolarizations (, top left). Overall, inactivation was accelerated through the reduction of time constants for both components at triggering voltages of 0 and 10 mV and enhancement of the fast fraction at 10 to 30 mV (). Finally, halothane effects on both components of α^1Cβ^2aα²/δ¹ inactivation were enhanced by concentration ().

The results demonstrate that halothane differentially modulated currents through recombinant LCCs with divergent gating characteristics determined by α²/δ¹ coexpression. Halothane depressed I^peak and enhanced apparent inactivation for both channels. The magnitude of these effects was markedly greater with coexpression of α²/δ¹ subunit, which also enhanced voltage-dependent inactivation. A central result is that halothane depression of transmembrane charge transfer and mean open probability is primarily governed by reductions in I^peak and not by changes in current time course (activation and inactivation). Furthermore, depression of I^peak arises from channel transitions to nonactivatable states occurring at resting membrane potential or early in the activation cascade. The findings provide strong evidence for the hypothesis that the phenotypic differences associated with the changes in voltage-dependent inactivation play key roles in the mechanism of inhibition of LCCs by halothane. However, it remains unclear as to whether the important effects of halothane on I^peak arise from differences in voltage-dependent inactivation or other attendant but unrecognized changes in phenotype resulting from α²/δ¹ coexpression. Overall, these results set the stage for studies to elucidate critical channel structures involved in the halothane inhibition of cardiac LCCs and to provide a deeper understanding of the inhibitory mechanism.

Coexpression of α²/δ¹ subunit with the pore-forming α subunit and accessory β subunit enhances macroscopic inactivation.17,,19 As a result, we studied channels arising from α^1C and β^2a subunits coexpressed with and without the α²/δ¹ subunit. Coexpression of α²/δ¹ subunit resulted in macroscopic currents with enhanced inactivation, and leftward shifts of I-V and conductance–voltage relations consistent with previous reports. Reproduction of these features also supports the position that green fluorescent protein tagging of the α^1C subunit does not alter fundamental channel function. Halothane produced reversible, concentration-dependent depression of I^peak for both channels (). Concentration–response relations for I^peak are reported for a triggering potential of 10 mV, at which the halothane effects on these endpoints reached a plateau as a function of activating voltage (), suggesting that differences in activation did not contribute to the findings.

α^1Cβ^2aα²/δ¹ likely reflects the function of in vivo  channels. Therefore, it is reasonable to compare the α^1Cβ^2aα²/δ¹ results from this study with those reported for native LCCs. Halothane (0.4 mm) depressed I^peak of α^1Cβ^2aα²/δ¹ by 34%, which is roughly comparable to the 25–45% I^peak reduction reported for native LCCs with Ba²⁺ as the charge carrier: I^peak was reduced 25% by 0.45 mm halothane in guinea pig ventricular myocytes, by 35% with 0.4 mm halothane in canine ventricular myocytes, by 35% with 0.45 mm halothane in canine cardiac Purkinje cells, and by 25% with 0.41 mm halothane in neuronal SH-SY5Y cells. Similar results have been reported with recombinant α^1Cβ^2bα²/δ¹ channels expressed in Xenopus  oocytes, where 0.59 mm halothane depressed I^peak by 45%.

The results of the current study suggest that halothane depresses I^peak through the reduction of activatable channels at resting membrane potentials. However, the underlying mechanism remains unclear but may be explained by a hyperpolarizing shift in the steady state inactivation. Isoflurane has been reported to produce significant shifts in the steady state inactivation curve in recombinant α^1Eβ^2bα²/δ¹ and in native LCCs in cardiac and nervous tissue.5,,24,,25,,26,,27 However, such shifts have not been reported with halothane in native cardiac5,,28 or recombinant LCCs, pointing to possible divergent effects of these volatile agents on LCC function. Examination of steady state inactivation was beyond the scope of the current study. Interestingly, single-channel studies have demonstrated that halothane significantly increases the fraction of null sweeps and the duration of sojourns into nonconducting states.5,,7 In the absence of hyperpolarizing shifts of steady state inactivation, these single-channel results in combination with unaltered macroscopic activation suggest that halothane depresses I^peak by promoting transitions from resting to drug-induced nonactivatable states as suggested by Pancrazio. Overall, we interpret these findings as support for distinct but overlapping mechanisms of LCC inhibition by halothane and isoflurane as suggested previously.1,,5,,30 This concept is consistent with the structural diversity of these compounds that gives rise to marked differences in molecular polarity.

Reductions in Ca²⁺ entry through LCCs contribute importantly to halothane depression of the contractile state of the heart. To gain insight into halothane-mediated depression of Ca²⁺ entry, we computed charge transfer (Q^T) triggered by depolarizations (+10 mV, 300 ms) that roughly approximate the magnitude and duration of a cardiac action potential. Q^T is also proportional to mean open probability. The results indicate that changes in I^peak are the primary determinant of charge entry through LCCs with halothane. Halothane produced enhancement of inactivation during a triggering depolarization. However, the resultant current decrease due to inactivation contributed little to the reduction of Q^T and mean open probability relative to the effects of I^peak. It is possible that in vivo , the impact of reductions in I^peak could be magnified because inhibition of LCCs causes cardiac action potential shortening, which itself leads to additional decrease in Ca²⁺ entry. The results should be interpreted with some caution because Ba²⁺ was used as the charge carrier in this study. Reduction of I^peak was significantly greater for Ba²⁺versus  Ca²⁺ as the charge carrier (68 and 57%, respectively), but only at highest halothane concentration (1.8 mm) in cardiac myocytes. Overall, the results suggest that with regard to cardiac contractility, an important effect of halothane is depression of Ca²⁺ entry into cardiac myocytes through reduction of I^peak. Rapid activation relative to inactivation and conductance–voltage relations unchanged by halothane argue for the involvement of channels states present at resting membrane potentials in the mechanism of halothane depression of I^peak but do not rule out the involvement of states visited early in activation.

Coexpression of α²/δ¹ subunit enhanced inactivation as well as increased sensitivity to the effects of halothane on inactivation (). Divergent concentration–response relations in magnitude of inactivation (I³⁰⁰/I^peak) for the two types of channels were obtained using a triggering potential of +10 mV. Halothane effects on inactivation reached an apparent plateau at a triggering potential of +10 mV (4,,5,,6) despite distinctive voltage dependent gating for both of these two channels types (). Therefore, such dissimilarities in native gating of these channels are unlikely to contribute to the differences in halothane effects.

In the current study, Ba²⁺ was the charge carrier, and pipette solutions included a Ca²⁺ chelator, which minimized contributions by Ca²⁺-mediated current inactivation. Therefore, current inactivation is primarily due to a voltage-dependent process rather one triggered by Ca²⁺. Overall, these findings are interpreted to indicate that molecular transitions and or channel states involved in voltage-dependent inactivation play a role in the pharmacologic action of halothane as suggested by Pancrazio and has also been proposed for isoflurane. However, the above result strongly points to the possibility that unrecognized phenotypic changes associated with altered inactivation as induced by coexpression of α²/δ¹ may be importantly involved in the action of halothane. Possible phenotypic changes may include fundamental changes in binding site affinity or accessibility, or changes in the allosteric effects of binding site occupation independent of affinity changes, for example. Finally, the results suggest that the contribution of changes in apparent macroscopic inactivation assumes a role secondary to changes in I^peak in reducing Ca²⁺ entry.

Voltage-dependent inactivation is a prominent feature of α^1C and L-type calcium channels. Halothane increases the magnitude of inactivation and the apparent inactivation rate observed during 300-ms depolarizations. These effects were greater when accompanied by enhanced intrinsic voltage-dependent inactivation produced by coexpression with α²/δ¹ (2,,5,,6). For α^1Cβ^2aα²/δ¹ channels, 0.4 mm halothane produced roughly similar current reductions with 300 ms of depolarization in guinea pig myocytes and neuronal SH-SY5Y cells with Ba²⁺ as the charge carrier. Halothane did not enhance inactivation in recombinant α^1Cβ^2bα²/δ¹ channels expressed in Xenopus  oocytes. However, these channels also showed little inactivation in control conditions, which may be attributed to the heterologous expression system or accessory subunit subtypes. When Ca²⁺ is the charge carrier, halothane also enhanced inactivation in CA1 neurons and GH3 cells, which may also involve effects on Ca²⁺-mediated inactivation. However, Fassl investigated the effect of halothane on LCCs in human atrial cells and found slowing of inactivation at approximately 2 MAC. This latter result may reflect species or preparation differences or genuine divergence between atrial and ventricular channels to the effects of VA.

Halothane reduces the time to I^peak for both channels, which is accompanied by acceleration of macroscopic activation. The results are consistent with those in adrenal chromaffin cells, where 1.4 mm halothane decreased LCC calcium currents by approximately 30% and enhanced activation rate with no change in the voltage dependence of peak conductance. Overall, these observations may be explained by rapid activation relative to inactivation in combination with commensurate halothane-induced increases in microscopic activation and deactivation rates such that voltage dependence of peak open probability is unaltered. In contrast, halothane slowed activation in cultured neuronal SH-SY5Y cells and had no effect in rat sensory neurons and GH3 pituitary cells. These findings point to pharmacologic differences among these LCCs derived from different cells and preparations.

To explore for possible state dependence of LCC inhibition, we investigated the voltage dependence of halothane acceleration of inactivation. Triggering voltage determines peak open probability as reported by conductance–voltage relations (). To gain insight into the involvement of states associated with channel activation (open and inactivated), we investigated changes in current time course over a range of triggering potentials. Halothane accelerated inactivation kinetics of α^1Cβ^2a channels, but with little voltage dependence (). Both components of α^1Cβ^2aα²/δ¹ inactivation were accelerated by triggering voltages ranging from 0 to +10 mV, whereas fast fraction was enhanced at voltages exceeding 10 mV (). The results suggest that halothane acts on open and inactivated states of α^1Cβ^2aα²/δ¹ by either promotion of these states, leading to inactivation or direct inhibition.

The authors thank Robert Kass, Ph.D. (Professor of Pharmacology, Columbia University, New York, New York), for rabbit skeletal muscle α²/δ¹ and heart β^2a and Robert Dirksen, Ph.D. (Associate Professor of Pharmacology and Physiology, University of Rochester, Rochester, New York), for α^1C tagged with green fluorescent protein.