Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation.
HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy.
Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels.
The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.
VOLATILE anesthetics (VAs) depress cardiac contractility by reducing cytosolic Ca2+ concentration through alteration of cellular mechanisms governing Ca2+ homeostasis. The critical first step in the increase of cytosolic Ca2+ in cardiac excitation–contraction coupling is the transmembrane entry of Ca2+ mediated by the opening of L-type Ca2+ channels (LCCs) triggered by membrane depolarization. The VA halothane is known to inhibit the entry of Ca2+ through the LCCs, which seems to be a primary effect by which halothane depresses cardiac contractility.1,,2,,3,,4
Volatile anesthetics seem to depress LCC activity and subsequent Ca2+ entry by enhancing apparent channel inactivation.5,,6,,7,,8,,9,,10 However, drug block of open channels may also manifest as enhancement of apparent inactivation, and recovery from drug-blocked states may mimic delayed recovery from inactivated states. Therefore, it is plausible that the apparent VA effects on inactivation may actually arise from a mechanism involving VA inhibition of open channels as reported in other examples of drug inhibition of ion channels.11,,12,,13
L-type Ca2+ channels are composed of multiple protein subunits (α, β, α2/δ, and γ) where the α subunit contains the pore forming region. The α subunit is composed of four domains, each with six transmembrane segments. Auxiliary β and α2/δ subunits modulate the kinetics of channel opening as well as increase the number of functional channels expressed in heterologous expression systems such as Xenopus oocytes and human embryonic kidney cells (HEK-293). Channel inactivation is considered to have two distinct components that are each dependent on membrane voltage or Ca2+ influx. Coexpression of the α2/δ subunit enhances voltage-dependent inactivation of αβ channels.16,,17
The strategy of this study was to manipulate channel phenotype using recombinant channels of varying subunit composition. In addition, heterologous protein expression used small cells from mammalian cell lines that provided for high resolution analysis of current time courses. The objectives of this study were (1) to investigate the role of voltage-dependent inactivation in halothane inhibition of cardiac LCCs carried by recombinant α1Cβ2 and α1Cβ2α2/δ1 channels and (2) to determine the effects of halothane on the time course of the voltage-triggered activity in recombinant channels expressed in HEK-293 cells. Notably, this study is the first to investigate VA effects on recombinant LCCs expressed in a human cell line. Coexpression of α2/δ1 subunit with α1Cβ2a (CaV1.2) resulted in enhanced inactivation of Ba2+ currents as previously described and greater inactivation enhancement and inhibition of peak currents (Ipeak). Time course analysis indicates that halothane accelerated activation kinetics in both channels unattended by significant changes in peak conductance. Halothane inhibition of Ipeak arises from channel transitions to nonactivatable states occurring at resting membrane potentials or early in activation, and primarily governs reductions in α1Cβ2aα2/δ1 transmembrane charge transfer thought to heavily influence depression of cardiac contractile state.
Materials and Methods
HEK-293 cells were transiently cotransfected by calcium phosphate precipitation with complementary DNAs encoding α1C tagged with green fluorescent protein on the N termini to serve as a reporter, and rabbit heart β2a, with and without rabbit skeletal muscle α2/δ1 subunits. Cells were initially cotransfected with α1C, but this method yielded low transfection efficiencies (< 5%) leading to poor experimental efficiency. To facilitate the identification of cells expressing α1C, transfections were conducted with α1C 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 α1C 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 (Gpeak) was calculated using
where V is the voltage of the activating depolarization, Ipeak is the current maximum during the activating depolarization, and Vrev 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 Gpeak normalized by maximum Gpeak 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, V1/2 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 τa, τb, and τc, 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 Calculation
Transmembrane charge transfer (QT) 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 Ipeak changes were approximated by calculating fractional Ipeak reduction relative to control. This approach determines QT occurring in the absence of alterations in current time course caused by changes in activation and inactivation kinetics. Reductions in QT that were unaccounted for by changes in Ipeak were attributed to changes in gating kinetics reflected in alterations in current time course. Relative contributions of Ipeak and kinetics were then calculated straightforwardly, assuming these two represented all contributing factors.
Currents from Cells Transfected with α1Cβ2a or α1Cβ2aα2/δ1 Subunits
shows a representative family of current responses triggered by a range of voltage steps from single HEK-293 cells transfected with either α1Cβ2a or α1Cβ2aα2/δ1 subunits. The peak I-V relations () show that maximum Ipeak was evoked at approximately +20 mV for α1Cβ2a channels, whereas that for α1Cβ2aα2/δ1 channels is leftward shifted by approximately 10 mV, consistent with previous reports. Coexpression of α2/δ1 with α1Cβ2a 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.
Halothane Depression of Ipeak and Inactivation Enhancement Are Potentiated by α2/δ1 Coexpression
We next investigated the concentration dependence of halothane depression of Ipeak 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 Ipeak and altered the time course of currents elicited from individual cells expressing α1Cβ2a and α1Cβ2aα2/δ1 channels (, top). The magnitude of Ipeak depression was confirmed in grouped data and over a range of halothane concentrations (, left). Responses normalized to Ipeak 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 Ipeak, and therefore, I300 was plotted after normalization for Ipeak (I300/Ipeak; , right). These relations show that effects on inactivation of α1Cβ2aα2/δ1 are markedly greater than α1Cβ2a 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 Ipeak 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 Ipeak. Overall, the results suggest that phenotypic changes associated with α2/δ1 coexpression potentiate halothane effects on Ipeak and apparent inactivation and are consistent with the view that voltage-dependent inactivation is a target of VA action.
Halothane Depression of Transmembrane Charge Transfer and Mean Open Probability Arise from Ipeak Reduction
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 (Ipeak) and time course (gating kinetics), which are both affected by halothane (). We investigated the impact of changes in Ipeak and kinetics on computed transmembrane charge transfer (QT; 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 (QT) during a triggering stimulus is proportional to the area under the current time course (hatched area). Halothane (1 mm) markedly reduced QT in both channels. Charge transferred (QT) was steeply dependent on halothane concentration for both channels (, left). Approximately 1½ minimum alveolar concentration (MAC) (approximately 0.6 mm) of halothane depressed QT by nearly 50% in α1Cβ2aα2/δ1 channels, whereas a similar effect in α1Cβ2a channels required more than threefold greater halothane concentration (> 2 mm). Changes in Ipeak accounted for more than 75% of the change in QT 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 Ipeak (, bottom). The similarity of Ipeak relations despite clear differences in inactivation kinetics between these channels strongly supports a primary role of Ipeak in determining charge transfer. QT was computed over a period of constant membrane potential (+10 mV, 300 ms). Mean open channel probability is proportional to QT, 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 Ipeak plays a primary role in the reduction of QT 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 QT 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 Ipeak.
Halothane Effects on Current– and Conductance–Voltage Relations
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 Ipeak 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 (Gpeak)–voltage relations. Gpeak reports the peak channel open probability. Gpeak 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 Gpeak, 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 Ipeak (). 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 ().
Halothane Alters Activation and Inactivation Kinetics
Normalized current traces indicate that halothane reduced the time to Ipeak for both channels (, bottom, insets). Time to Ipeak, determined by inspection, was reduced by halothane for α1Cβ2a (control, 54.8 ± 6.9 ms; halothane, 37.0 ± 4.3 ms; P < 0.01) and α1Cβ2aα2/δ1 (control, 21.8 ± 3.7 ms; halothane, 14.1 ± 2.3 ms; P < 0.01). Gpeak 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 Ipeak 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 α1Cβ2a current time course was well fit by a triexponential function (equation 3), where activation was described by two exponential components (time constants: fast [τf] and slow [τs]) 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 α1Cβ2aα2/δ1 were also well fit by a triexponential function (equation 3) but with monoexponential activation (time constant: τa) and biexponential inactivation (time constants: τs and τf) (), 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 α1Cβ2aα2/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 Ipeak 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 Ipeak and not by changes in current time course (activation and inactivation). Furthermore, depression of Ipeak 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 Ipeak 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 Potentiates Inactivation and Halothane Inhibition of Ipeak
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 α1C and β2a 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 α1C subunit does not alter fundamental channel function. Halothane produced reversible, concentration-dependent depression of Ipeak for both channels (). Concentration–response relations for Ipeak 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.
α1Cβ2aα2/δ1 likely reflects the function of in vivo channels. Therefore, it is reasonable to compare the α1Cβ2aα2/δ1 results from this study with those reported for native LCCs. Halothane (0.4 mm) depressed Ipeak of α1Cβ2aα2/δ1 by 34%, which is roughly comparable to the 25–45% Ipeak reduction reported for native LCCs with Ba2+ as the charge carrier: Ipeak 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 α1Cβ2bα2/δ1 channels expressed in Xenopus oocytes, where 0.59 mm halothane depressed Ipeak by 45%.
The results of the current study suggest that halothane depresses Ipeak 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 α1Eβ2bα2/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 Ipeak 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.
Changes in Ipeak Determine Reductions in Charge Transfer and Mean Open Probability Produced by Halothane
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 (QT) triggered by depolarizations (+10 mV, 300 ms) that roughly approximate the magnitude and duration of a cardiac action potential. QT is also proportional to mean open probability. The results indicate that changes in Ipeak 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 QT and mean open probability relative to the effects of Ipeak. It is possible that in vivo , the impact of reductions in Ipeak 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 Ipeak 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 Ipeak. 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 Ipeak but do not rule out the involvement of states visited early in activation.
Coexpression of α2/δ1 Potentiates Inactivation and Enhancement by Halothane
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/Ipeak) 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 Ipeak in reducing Ca2+ entry.
Voltage-dependent inactivation is a prominent feature of α1C 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 α1Cβ2aα2/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 α1Cβ2bα2/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 Accelerates Activation
Halothane reduces the time to Ipeak 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.
Halothane Acceleration of α1Cβ2aα2/δ1 Inactivation Kinetics Is Voltage Dependent
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 α1Cβ2a channels, but with little voltage dependence (). Both components of α1Cβ2aα2/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 α1Cβ2aα2/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 β2a and Robert Dirksen, Ph.D. (Associate Professor of Pharmacology and Physiology, University of Rochester, Rochester, New York), for α1C tagged with green fluorescent protein.