Voltage-dependent inactivation of L-type Ca²⁺ currents in guinea-pig ventricular myocytes

Abstract

The objective of this study was to describe the kinetics of voltage-dependent inactivation of native cardiac L-type Ca²⁺ currents. Whole-cell currents were recorded from guinea-pig isolated ventricular myocytes. Voltage-dependent inactivation was separated from Ca²⁺-dependent inactivation by replacing extracellular Ca²⁺ with Mg²⁺ and recording outward currents through Ca²⁺ channels. Voltage-dependent inactivation accelerated from slow monophasic decay at −30 mV to maximal rapid biphasic decay at +20 mV. Maximal voltage-dependent inactivation occurred with τ_f ≈30 ms and τ_s ≈300 ms, the fast component of decay accounted for 70 % of the current amplitude. In basal conditions Ca²⁺ current availability was sigmoid. Isoproterenol (isoprenaline) evoked a large increase in a time-independent component of the Ca²⁺ current which also increased with depolarisation. This was responsible for the apparent recovery of Ca²⁺ channel current availability at positive membrane potentials and thus a U-shaped availability-voltage (A-V) relationship. It is concluded that β-adrenergic stimulation altered the reaction of native cardiac L-type Ca²⁺ channels to membrane voltage. In basal conditions, voltage accelerated inactivation. In isoproterenol, voltage could also reduce inactivation.

A key to understanding the contribution of L-type Ca²⁺ currents (I_Ca,L) to the cardiac action potential and the process of excitation-contraction coupling is the mechanism of inactivation. Inactivation of L-type Ca²⁺ channels proceeds according to voltage- and Ca²⁺-dependent mechanisms (Pelzer et al. 1990). Recently a great deal of interest has centred upon the mechanism of Ca²⁺-induced inactivation with the implication of the association of the ubiquitous Ca²⁺-binding protein calmodulin with the intracellular C-terminus of the α_1C subunit of the Ca_v1.2 channel (see Anderson, 2001 for recent review). The molecular basis of voltage-dependent inactivation of Ca²⁺ channels has also been extensively studied (reviewed in Stotz & Zamponi, 2001). In comparison with the dynamic regulation of inactivation induced by Ca²⁺ (Hamilton et al. 2000; Anderson, 2001; Bers, 2002), inactivation due to voltage has generally been considered to play a minor role in the decay of L-type Ca²⁺ channel currents in cardiac myocytes (Bechem & Pott, 1985; Imredy & Yue 1994; Linz & Meyer, 1998; Puglishi et al. 1999; Sun et al. 2000). Recent studies challenge this view (Mitarai et al. 2000; Findlay, 2002a,b).

Mitarai et al. (2000) provided the first clear evidence that β-adrenergic stimulation could profoundly affect the character of voltage-dependent inactivation of native cardiac L-type Ca²⁺ channels. They suggested that the apparent slowing of the voltage-dependent decay of Ca²⁺ channel currents induced by isoproterenol (isoprenaline) (Bean et al. 1984; Tsien et al. 1986; Tiaho et al. 1991; Findlay, 2002a) resulted from the conversion of channels from a rapidly inactivating to a slowly inactivating kinetic form. The consequence of this result is to suggest that models of cardiac muscle Ca²⁺ currents which have been developed to describe their behaviour under basal conditions, but which evaluated voltage-dependent decay following β-adrenergic stimulation, will have underestimated the contribution of voltage (Linz & Meyer, 1998; Puglisi et al. 1999; Sun et al. 2000). The general consensus of opinion which described the primary contribution of Ca²⁺-induced inactivation to the decay of Ca²⁺ currents will also have been overestimated since this also was measured under experimental conditions which were the equivalent of β-adrenergic stimulation (Bechem & Pott, 1985; Imredy & Yue, 1994). Thus a previous investigation from this laboratory found that whereas following β-adrenergic stimulation the kinetics of inactivation of the Ca²⁺ current were almost exclusively dependent upon Ca²⁺, under basal conditions a large part of inactivation was independent of Ca²⁺ (Findlay, 2002b). These results revealed the major contribution of voltage-dependent inactivation to the decay of L-type Ca²⁺ currents under basal conditions. That study examined voltage-dependent inactivation at one membrane voltage (+10 mV) in order to optimally separate the contributions of Ca²⁺ and voltage. The objective of the present study was to examine the process of voltage-dependent inactivation in native cardiac L-type Ca²⁺ channels over a more extensive range of membrane voltages. This information may then be used to develop models of the Ca²⁺ current and cardiac action potential which take full account of the contribution of voltage-dependent inactivation.

This investigation also addressed a second question. Previous studies have shown that under basal conditions the availability-voltage (A-V) relationship for I_Ca,L recorded in the absence of extracellular Ca²⁺ was sigmoid (Hadley & Hume, 1987; Findlay, 2002b). This suggested that under those conditions inactivation occurred via a voltage-dependent process with no interference from an ion-dependent inactivation mechanism (Ferreira et al. 1997; Findlay, 2002a). On the other hand, under the same experimental conditions following maximal β-adrenergic stimulation the A-V relationship was U-shaped with a clear ‘recovery’ of I_Ca,L following voltage-clamp steps to positive membrane potentials (Findlay, 2002b). Here it is shown that β-adrenergic stimulation resulted in a paradox where membrane voltage, rather than inducing inactivation, could induce a non-inactivating kinetic form of the channel. This mechanism could explain how a U-shaped relationship between inactivation and membrane voltage could arise in the absence of Ca²⁺-induced inactivation.

Methods

Cell preparation

All animal experiments were conducted according to the ethical standards of the Ministère Français de l'Agriculture (Licence number B37-261-4). Male guinea-pigs (250-400 g; purchased from Iffa-Credo Ltd, L'Abresle, France) were killed by cervical dislocation and the hearts were removed. Single ventricular myocytes were isolated using collagenase and protease digestion as described elsewhere (Le Guennec et al. 1993). Myocytes isolated from the left ventricle were aliquoted into 35 mm diameter plastic Petri dishes which served as experimental chambers. The storage solution consisted of the standard extracellular solution described below. Dishes which contained myocytes were kept on the laboratory bench and used within 6-8 h after isolation. Plastic Petri dishes which contained isolated myocytes were placed upon the stage of an Olympus CK2 inverted microscope. Isolated myocytes were superfused with experimental solutions via a parallel pipes system lowered into the vicinity of the cells. Fluid flow was maintained by gravity from syringe barrel reservoirs and the exchange of solutions was achieved by manual displacement of the pipes. Solution exchange around the myocyte was estimated to be complete in 5-6 s. All experiments were conducted at room temperature (≈23 °C) in a temperature-controlled laboratory.

Experimental procedures

Whole-cell current voltage-clamp experiments (Hamill et al. 1981) were conducted with an Axon Instruments 202A patch-clamp amplifier in resistive feedback mode (Axon Instruments, CA, USA). Pipettes were fabricated from thin walled borosilicate glass capillary tubes (Clark Electromedical Instruments, Pangbourne, UK) with a Narishige PB7 double-stage puller (Narishige Instruments, Tokyo, Japan). Pipettes were coated with Sylgard (Dow Corning, MI, USA) and then heat polished. Finished pipettes had a resistance of < 2 MΩ when filled with standard intracellular solution.

Experimental voltage-clamp protocols and data acquisition were controlled with Acquis1 software (Dipsi Industrie, Chatillon, France) installed upon a 386-20 PC computer. Data were filtered at either 1 or 2 kHz and acquired at 2 or 5 kHz, respectively. Cell capacitance and series resistance were compensated (≈80 %) with the Axon Instruments amplifier. Cell currents are expressed as cell current density in picoamps per picofarad (pA pF⁻¹). Voltage-clamp protocols were delivered to the cells from the holding potential of −80 mV. Each voltage-clamp protocol was preceded by a voltage step to −50 mV for a period of 1000 ms. This prior voltage step served to inactivate any residual Na⁺ current remaining after the application of 10 μm TTX and to inactivate any T-type Ca²⁺ current in ventricular myocytes of the guinea-pig (Balke et al. 1992). This study utilised two voltage-clamp protocols. The first was a double-pulse protocol which consisted of pre-pulses to between −50 and +80 mV in 10 mV increments, a 10 ms interval at −50 mV and a test pulse voltage step to +80 mV. The duration of the pre-pulses was either 1000 or 5000 ms. Current-voltage (I-V) relationships and the kinetics of decay of I_Ca,L were evaluated from currents recorded during pre-pulse voltage steps. Availability-voltage (A-V) relationships were obtained by measuring the amplitude of the current evoked by the test pulse voltage step and normalising these data to that recorded following a pre-pulse voltage step to −50 mV. The second voltage-clamp protocol was an envelope-type of double-pulse protocol where the test pulse voltage step to +80 mV was preceded by voltage steps to a given voltage for durations which varied between 10 and 1000 ms (see Fig. 1). This protocol was used to follow the time course of the development of inactivation from the progressive loss of availability of I_Ca,L. Data analysis was performed with Acquis1 and Origin 4.1 (Microcal Software, MA, USA). The Boltzmann function (Origin 4.1) was fitted to values between the maximum and minimum of normalised data values for the A-V relationships. All results are presented as mean ± s.e.m. values of data obtained from n different myocytes.

Figure 1. The time course of the development of voltage-dependent inactivation.

These normalised cell current traces were recorded from one representative isolated ventricular myocyte during pre-pulse voltage steps to −20 mV (A) and +10 mV (B) using the voltage-clamp protocol indicated schematically above the traces. For clarity only four traces representing currents recorded with no pre-pulse and pre-pulses of 50, 200 and 1000 ms duration (arrows) are shown here. The time scale is indicated by the horizontal bar in A. The dashed lines indicate the zero current level. Cell currents were recorded in the absence of extracellular Ca²⁺.

Experimental solutions

The standard extracellular solution which was used to fill the Petri dishes and store myocytes prior to experiments contained (mm): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, 10 Hepes (pH 7.4 with NaOH). Tetrodotoxin (TTX citrate salt, 10 μm, from either Alomone Labs, Jerusalem, Israel or Latoxan, Valence, France) was added to this standard solution when it was used to superfuse cells during experiments. Calcium-free (zero calcium) extracellular solution contained 250 μm EGTA-NaOH, 3 mm MgCl₂ and no added Ca²⁺. The standard intracellular solution which was used to fill the patch pipettes contained (mm): 140 KCl, 10 NaCl, 5 EGTA-KOH, 1.4 MgCl₂, 0.1 CaCl₂, 2 ATP-Mg²⁺, 10 glucose, 10 Hepes (pH 7.3 with KOH). The estimated free concentrations of Mg²⁺ and Ca²⁺ in this solution were 1 mm and 1 nm, respectively. In experiments where β-adrenergic stimulation was to be applied to the myocytes, the pipette solutions also contained 100 μm GTP. Isoproterenol was prepared daily as a 100 μm stock solution in distilled water and added to extracellular solutions to give a final concentration of 100 nm. Ryanodine was prepared as a 1 mm stock solution in distilled water and added to extracellular solutions to give a final concentration of 5 μm. Chemicals were purchsed from Sigma-Aldrich.

Voltage-gated currents through L-type Ca²⁺ channels were extracted from the ensemble whole-cell currents with 200 μm CdCl₂. The limitations of this method are fully discussed by Linz & Meyer (1998). The cell current records obtained in the presence of Cd²⁺ were digitally subtracted from those recorded in the absence of Cd²⁺ with Acquis1.

Results

The characteristics of the L-type Ca²⁺ channel current which is recorded in isolated ventricular myocytes of the guinea-pig in the absence of extracellular Ca²⁺ have been described in Findlay (2002b). The results shown in this study were all obtained when myocytes were bathed in the zero calcium solution, which was described in Methods. It should be noted that the exposure of the myocytes to this solution was limited to the time necessary to achieve the voltage-clamp protocol; otherwise they were superfused with the normal extracellular solution which contained 2 mm Ca²⁺.

Voltage-dependent inactivation of L-type Ca²⁺ channels

This series of experiments sought to examine the time course of the development of voltage-dependent inactivation over a range of physiologically important membrane potentials. Since, between membrane potentials of −30 and +10 mV no current flowed through Ca²⁺ channels when Mg²⁺ had replaced Ca²⁺ in the extracellular medium (Fukushima & Hagiwara, 1985; Hess et al. 1986; Hadley & Hume, 1987; Findlay, 2002b), the inactivation of I_Ca,L was assessed indirectly by monitoring the availability of the outward Ca²⁺ channel current evoked by a voltage step to +80 mV (Hadley & Hume, 1987). Inactivation was induced by pre-pulse voltage steps of variable duration (Findlay, 2002b). Figure 1A illustrates the response of an isolated myocyte to pre-pulse voltage steps to −20 mV of 50, 200 and 1000 ms duration. Elongation of the pre-pulse, which evoked no current flow through Ca²⁺ channels, resulted in the progressive reduction of the amplitude of the outward Ca²⁺ channel current evoked by the test pulse to +80 mV. Pre-pulses to +10 mV provoked a more rapid and more extensive reduction of the test pulse current amplitude (Fig. 1B).

The effect of membrane potential upon the rate of inactivation of I_Ca,L recorded in the absence of Ca²⁺ is shown in Fig. 2A. The time course of the development of inactivation at each voltage could be fitted by exponential functions (lines in Fig. 2A) whose time constants are shown in Fig. 3A and B. The rate of inactivation accelerated with depolarisation of the membrane potential from −30 to ≈+20 mV. Voltage above ≈+20 mV had little or no further effect. The rate of inactivation recorded at −30 and −20 mV could be described by a single slow exponential function. The rate of inactivation recorded at −10 mV and more positive voltages required a double-exponential function. Maximal inactivation was biphasic with time constants of ≈30 and ≈300 ms. Figure 2B shows that a single and voltage-dependent process could account for these results, since, when the data from Fig. 2A were displayed upon a voltage rather than a time axis, a Boltzmann relation could be fitted to the data irrespective of the duration of the pre-pulse voltage step.

Figure 2. Exponential development of voltage-dependent inactivation.

A, the time course of voltage-dependent inactivation of I_Ca,L at different membrane potentials. Lines represent the fitting of either a single or a double-exponential function to the data. B, the effect of pre-pulse duration upon the A-V relationship of the Ca²⁺ channel current. The data from A have been transposed onto a voltage scale. The lines represent fits of the Boltzmann function to these data which correspond to I_Ca,L availability recorded following pre-pulse steps of 10, 20, 50, 100, 200, 500 and 1000 ms duration reading from top to bottom. A and B, ▪, pre-pulse voltage (pp) −30 mV (n = 11). •, pp −20 mV (n = 19); ▴, pp −10 mV (n = 11); ▾, pp 0 mV (n = 11); ♦, pp +10 mV (n = 20); □, pp +20 mV (n = 12); ○, pp +30 mV (n = 11); ▵, pp +40 mV (n = 16); ▿, pp +80 mV (n = 13).

Figure 3. Voltage-dependent inactivation and membrane potential.

Analysis of the development of voltage-dependent inactivation was extracted from the details of the exponential functions fitted to the data in Fig. 2A. A, the fast time constant. B, the slow time constant. C, the initial amplitude of each component of either a single exponential function (slow decline (τ) and offset for data recorded following pre-pulse steps to −30 and −20 mV) or a double-exponential function (fast decline (τ_f), slow decline (τ_s) and offset for data recorded following pre-pulse steps to between −10 and +40 mV) was determined and expressed relative to their respective totals.

The inactivation of I_Ca,L in cardiac myocytes is generally considered to follow a biphasic time course because of the joint influence of voltage- and Ca²⁺-dependent inactivation (Pelzer et al. 1990). On the other hand, the results shown in Fig. 2 (and see also Mitarai et al. 2000; Findlay, 2002b) indicate that I_Ca,L could inactivate with a biphasic time course in the absence of Ca²⁺-dependent inactivation. This observation led Mitarai et al. (2000) to consider not only the time course of inactivation but also what proportions of the whole-cell current corresponded to such kinetically distinct components. The single exponential development of inactivation which occurred at −30 and −20 mV suggested that at these voltages whole-cell I_Ca,L would be composed of ion channels which either inactivated in a slow time-dependent manner or which showed no time-dependent behaviour on this time scale. The double-exponential development of inactivation which occurred at −10 mV suggested that some ion channels had now adopted a rapid form of time-dependent inactivation. These distinct kinetic forms of the L-type Ca²⁺ channel co-existed within the whole-cell population of channels and Fig. 3C shows that, from the starting point of −30 mV, depolarisation was associated with the progressive reduction of the time-independent component, at first the amplitude of the slow time-dependent component increased then declined, and the rapid time-dependent component first appeared at −10 mV and progressively increased. A steady-state was reached at +20 mV and further depolarisation had no further effect upon the distribution of ion channels between the different kinetic forms of behaviour within the whole-cell population.

The results shown in Figs 1–3 define the voltage-dependent behaviour of L-type Ca²⁺ channels over the physiologically important range of membrane potentials. Several conclusions could be drawn from this data. A voltage-dependent process was responsible for the graded time-dependent development of inactivation (Fig. 2B). The overall kinetic of inactivation was dependent upon the membrane potential (Fig. 1 and Fig. 3A and B). Individual L-type Ca²⁺ channels could be assigned to one of the three distinct kinetic groups (Fig. 3C). Membrane potential determined the distribution of ion channels between these kinetic groups (Fig. 3C). Maximal voltage-dependent inactivation was represented by ≈70 % of the channel population showing rapid inactivation with a time constant of ≈30 ms.

Figure 4 illustrates a similar analysis of the time course of the development of inactivation here applied to the total inactivation of I_Ca,L, which was recorded in the presence of extracellular Ca²⁺. The inactivation of I_Ca,L was recorded directly from the decay of inward Ca²⁺ currents. To allow a certain degree of comparison between these data and those of isolated voltage-dependent inactivation (Fig. 2 and Fig. 3), these experiments were conducted in the presence of 5 μm ryanodine in order to block Ca²⁺ release from the sarcoplasmic reticulum (Ca²⁺-induced Ca²⁺ release, CICR). The isolation of voltage-dependent inactivation by the exclusion of extracellular Ca²⁺ precluded a contribution of CICR to the time course of inactivation (Findlay, 2002b). In the context of this study it was therefore appropriate to also exclude the contribution of CICR to the total inactivation of I_Ca,L (Adachi-Akahane et al. 1996; Sham, 1997).

Figure 4. The time course of the development of total inactivation of I_Ca,L.

I_Ca,L was evoked by 1000 ms duration voltage-clamp steps to between −30 and +30 mV in myocytes which were bathed in extracellular solution which contained 2 mm Ca²⁺ and 5 μm ryanodine (n = 36). The inactivation of I_Ca,L was evaluated by measuring the amplitude of the inward Ca²⁺ current 10, 20, 50, 100, 200, 500 and 1000 ms following the onset of the voltage step. These values were then normalised to the amplitude of the peak inward current. ▪, voltage step to −30 mV; •, −20 mV; ▵, −10 mV; ▾, 0 mV; □, +10 mV; ○, +20 mV; ▿, +30 mV. The lines represent fits of double-exponential functions to the data.

By comparison with the data which described isolated voltage-dependent inactivation (Fig. 2A), two main points arose from the analysis of total inactivation (Fig. 4). First, the time course of decay of I_Ca,L recorded at −30 and −20 mV was described by double-exponential functions. Second, the rate of decay which increased with depolarisation to be maximal between 0 and +10 mV was then apparently slower at more positive voltages. Thus at negative membrane potentials a fast component of inactivation was visible in total inactivation (Fig. 5) which was absent from the isolated voltage-dependent inactivation (Fig. 3). Maximal inactivation at ≈+10 mV for voltage-dependent (Fig. 3) and total inactivation (Fig. 5) showed great similarity, which is in line with previous observations (Findlay, 2002a,b), though the amplitude of the component of fast inactivation was relatively greater in Ca²⁺ (Fig. 5C) than in its absence (Fig. 3C). At more positive membrane potentials certain differences were apparent. Thus total inactivation showed a reduction of the contribution of the component with rapid inactivation (Fig. 5C) and consequently an increase in the component with slow inactivation. The relationship between the kinetic components of isolated voltage-dependent inactivation were unchanged at these voltages (Fig. 3C).

Figure 5. Total inactivation of I_Ca,L and membrane potential.

Analysis of the development of voltage-dependent inactivation was extracted from the details of the exponential functions fitted to the data in Fig. 4. A, the fast time constant of inactivation. B, the slow time constant of inactivation. C, the proportion of the total current occupied by fast inactivation, slow inactivation and an offset.

These results can be understood, and differences between results obtained for isolated voltage-dependent inactivation and total inactivation accounted for, now that it has been shown that biphasic decay of I_Ca,L results not only from the separate processes of voltage- and Ca²⁺-dependent inactivation (Pelzer et al. 1990), but that voltage-dependent decay also showed a biphasic time course (Mitarai et al. 2000; Findlay, 2002a,b). Thus total inactivation is the complex result of the superposition of the kinetics of Ca²⁺-induced inactivation, which was simplified in this study by the exclusion of the contribution of CICR, upon the biphasic time course of decay already imposed by the kinetic separation of the voltage-dependent behaviour of individual ion channels.

Voltage-dependent potentiation of I_Ca,L

The second question to be addressed in this study concerned the observation that, while the A-V relationship of I_Ca,L recorded under basal conditions in the absence of extracellular Ca²⁺ was sigmoid in form, following β-adrenergic stimulation it was U-shaped (Findlay, 2002b and see Fig. 6B). In the absence of extracellular Ca²⁺ and in the absence of cation influx under these experimental conditions there was no reason to consider that β-adrenergic stimulation had induced an ion-dependent inactivation mechanism. The cause of the recovery of availability of I_Ca,L following pre-pulse voltage steps to positive membrane potentials was investigated with a double-pulse voltage-clamp protocol with 5000 ms pre-pulse voltage steps. This protocol was considerably longer than those applied by either Mitarai et al. (2000) or Findlay (2002b) and it was hoped that this would approach as closely as possible the steady-state condition.

Figure 6. The effect of isoproterenol upon voltage-dependent inactivation of Ca²⁺ channel currents.

Ca²⁺ channel currents were recorded in the absence of extracellular Ca²⁺ with the double-pulse voltage-clamp protocol with 5000 ms duration pre-pulse voltage steps. Experiments were conducted first under basal conditions (lefthand traces in A and filled symbols in B) and then in the presence of 100 nm isoproterenol (righthand traces in A and open symbols in B). A, an illustration of voltage-dependent inactivation and recovery. These normalised records each represent three superimposed cell currents which had been evoked by the double-pulse voltage-clamp protocol with pre-pulse voltage steps to −50, 0 and +80 mV. The voltage-clamp protocol is shown schematically above the traces. The amplitude of the current evoked by the test pulse voltage step to +80 mV following each pre-pulse (pp) is indicated by the arrows to the right of the traces. The time scale on the left applies to both traces and the dotted lines indicate the zero current level. B, A-V relationships recorded following 5000 ms pre-pulse voltage steps (n = 10). The lines represent Boltzmann functions with the following parameters: for control data, maximum 1.00, minimum 0.09, V_0.5 −24 mV, slope 4.9 mV; for data with isoproterenol, maximum 1.00, minimum 0.25, V_0.5 −29 mV, slope 5.5 mV.

The cell current records shown in Fig. 6A illustrate the effects of β-adrenergic stimulation upon the Ca²⁺ channel currents recorded in the absence of extracellular Ca²⁺. The most obvious point is that isoproterenol provoked a drastic slowing of the decay of these currents (Mitarai et al. 2000; Findlay, 2002b) which was also reflected in the reduction of the minimum of the A-V relationship recorded in isoproterenol (Fig. 6B). In basal conditions the A-V relationship was essentially sigmoid (Fig. 6B, filled symbols), while following β-adrenergic stimulation the A-V relationship was U-shaped (Fig. 6B, open symbols), with a minimum at ≈0 mV and recovery with further depolarisation. The Boltzmann relation provided a close fit to the data recorded under control conditions, with a V_0.5 of −24 mV; in isoproterenol the Boltzmann relation could only describe the descending limb of the relationship and evaluated a V_0.5 of −29 mV. The minima of the A-V relationships were 0.09 in basal conditions and 0.25 in isoproterenol. It was therefore clear that a U-shaped A-V relationship did not result from the premature evaluation of availability. In fact, the comparison of Fig. 3D (50 ms pre-pulse duration) and 3B (1000 ms pre-pulse duration) in Findlay (2002b) with Fig. 6B shown here with a 5000 ms pre-pulse duration would actually suggest that the reverse was true; the extent of recovery of I_Ca,L during pre-pulse voltage steps to positive membrane potentials increased with time.

In order to resolve the question of the U-shaped A-V relationship which was recorded in the presence of isoproterenol (Fig. 6B) it was decided to analyse the decay of Ca²⁺ channel currents evoked by voltage steps to between +30 and +80 mV (Fig. 7), since it was following these that the major part of the ‘recovery’ of channel availability occurred (Fig. 6B). Within this range of voltages, under basal conditions the fast time constant of decay was essentially voltage independent, declining only slightly between +30 and +80 mV and isoproterenol slightly increased this at each voltage (Fig. 7A). The slow time constant of decay was clearly voltage dependent under basal conditions, accelerating with depolarisation (Fig. 7B). Isoproterenol accelerated the slow time constant at lower voltages while having no effect at higher voltages; thus in isoproterenol the slow time constant of decay was voltage independent (Fig. 7B). At each voltage the amplitude of the fast component of decay was reduced by β-adrenergic stimulation (Fig. 7C, squares) while the amplitudes of the slow (Fig. 7D) and time-independent (Fig. 7C, triangles) components of decay were increased. The initial amplitudes of the distinct components of decay were expressed relative to the amplitude of the total current. Under basal conditions, the composition of the current recorded between +30 and +80 mV was voltage independent and dominated (≈70 %) by fast decay (Fig. 7E). In the presence of isoproterenol, fast decay was reduced to ≈20 % of the total and the composition of the current was voltage dependent, with depolarisation increasing the contribution of the time-independent component (Fig. 7F). The data shown in Fig. 7C show that the amplitude of the fast decay (squares) and time-independent (triangles) components of I_Ca,L were interchanged between basal conditions and stimulation of the current by isoproterenol.

Figure 7. The effects of isoproterenol upon the voltage-dependent kinetic components of I_Ca,L.

Cell currents had been evoked by 5000 ms duration voltage steps to between +30 and +80 mV (n = 10). The decay of each current was fitted with a double-exponential function. Filled symbols represent data obtained under basal conditions and open symbols represent data recorded following the application of 100 nm isoproterenol in the same cells. A, the fast time constant of inactivation. B, the slow time constant of inactivation. C and D, the initial amplitudes of the fast (C, squares), slow (D, circles) and offset (C, triangles) components of the Ca²⁺ channel currents. E and F, the initial amplitudes of the different components of decay were expressed relative to the total current recorded at each voltage under basal conditions (E) and in the presence of isoproterenol (F).

It was possible to draw some conclusions from the data shown in Fig. 6 and Fig. 7. Whilst under basal conditions the A-V relationship of the Ca²⁺ channel current could be adequately described by a single voltage-dependent process, while following β-adrenergic stimulation this was no longer possible (Fig. 6B). The rising phase of the β-adrenergic U-shaped A-V relationship was the reflection of the voltage-dependent increase in an inactivation resistant component of the Ca²⁺ channel current (Fig. 7F). Isoproterenol slowed the decay of Ca²⁺ channel currents, not by having a marked effect upon the time constants of decay (Fig. 7A and B), but by altering the composition of the whole-cell Ca²⁺ channel current (Fig. 7E and F, and see Mitarai et al. 2000; Findlay, 2002b). In absolute terms isoproterenol reduced the amount of current that decayed with a fast time course (Fig. 7C). This component which dominated the decay of Ca²⁺ channel currents under basal conditions (Fig. 7E) was replaced by Ca²⁺ channel currents which showed no inactivation upon this time scale (Fig. 7C).

Discussion

This study supports the suggestion that β-adrenergic stimulation profoundly influenced the manner in which the L-type Ca²⁺ channel reacted to membrane voltage (Mitarai et al. 2000; Findlay, 2002b). In basal conditions progressive depolarisation induced channels to adopt a rapid form of inactivation. β-Adrenergic stimulation was associated with the reduction of this form of inactivation and now progressive depolarisation induced channels to assume a form which did not show inactivation. The molecular basis of this switch in the response of the Ca_V1.2 channel to membrane voltage remains to be determined.

Visually, an unusual aspect of the results presented in this study concerns the representation of Ca²⁺ channel currents as the outward flow of intracellular monovalent cations which may be K⁺ or Cs⁺ (Findlay, 2002b; Macianskiene et al. 2002). One is more accustomed to observe Ca²⁺ current which flows from the extracellular environment into the cells. In physiological terms the L-type Ca²⁺ channel can be regarded as being inwardly rectified for the passage of Ca²⁺. There is a genuine reversal potential between Ca²⁺ influx and intracellular monovalent cation efflux (Tsien et al. 1987). That, when Mg²⁺ replaced extracellular Ca²⁺, outward currents through Ca²⁺ channels could be recorded from voltages as low as +20 mV resulted from the fact that, with a quasi-physiological cation gradient, monovalent cation permeation through Ca²⁺ channels had a reversal potential close to 0 mV (Tsien et al. 1987). The block of these channels by external Mg²⁺ is relieved by K⁺ or Cs⁺ efflux according to the voltage and the driving force (Fukushima & Hagiwara, 1985; Hess et al. 1986). This gave rise to outward currents through Ca²⁺ channels at voltages more positive than +10 mV, while no current flowed either inward or outward through the channels at voltages between −40 and +10 mV (Fig. 1).

The kinetics of voltage-dependent inactivation of I_Ca,L under basal conditions are complex. Notwithstanding that a single voltage-dependent process could describe the relationship between inactivation and membrane potential (Fig. 2B), the time course of inactivation was not monotonic and itself varied with the voltage (Fig. 2A). Analysis of the behaviour of single Ca²⁺ channel currents has shown that individual Ca²⁺ channels may adopt particular and distinct kinetic behaviours (Hess et al. 1984; Nowyck et al. 1985; Rose et al. 1992). Hess et al. (1984) described these as ‘modes’. The point of this concept is that, without changing conditions, for example when applying repetitively a given voltage step, a channel may switch apparently spontaneously from one to another particular pattern of opening and closing. The application of different stimuli such as more or less depolarised voltage steps or the application of an agonist may privilege the adoption of a particular kinetic pattern. But it should be emphasised that this would not be to the exclusion of other kinetic patterns or prevent an ion channel from switching between kinetic patterns, though the probability of such a switch might be reduced. The consequence of this behaviour is that the whole-cell current may show complex kinetics due to the fact that the behaviour of its component ion channels is not evenly distributed around one type of kinetic behaviour but divided between a number of different kinetic patterns. In this context therefore the effect of voltage upon the kinetics of inactivation of I_Ca,L may be seen to lie less in the time constant(s) of the process (Fig. 3A and B) and more in the re-distribution of ion channels between different kinetic behaviours or ‘modes’ (Fig. 3C). Models of the L-type Ca²⁺ channel current need to take account of this diversity of kinetic behaviour and its modulation by membrane potential.

Mitarai et al. (2000) suggested that the effect of β-adrenergic stimulation upon the voltage-dependent inactivation of I_Ca,L resulted from the shift of the majority of Ca²⁺ channels from a rapidly to a slowly inactivating form. This study suggests rather that the exchange might be between Ca²⁺ channels which inactivate rapidly and those which do not inactivate on this time scale (Fig. 7C). The absence of a time-independent component of I_Ca,L from the analysis of Mitarai et al. (2000) might have been the consequence of their applying 300 ms duration voltage steps. On this short time scale it would be difficult to illustrate a steady-state condition. Nevertheless this study did confirm the observation of Mitarai et al. (2000) that β-adrenergic stimulation provoked the loss of ion channels which showed rapid inactivation. Isoproterenol also resulted in the conversion of the sigmoid voltage-dependent A-V relationship, which was recorded under basal conditions, into a U-shaped relationship, even in the absence of Ca²⁺-dependent inactivation (Findlay, 2002b). It was shown that this resulted from the fact that, with β-adrenergic stimulation, membrane voltage, rather than inducing inactivation, actually promoted a non-inactivating kinetic form of the channel (Fig. 7F). Pietrobon & Hess (1990) and Hirano et al. (1999) both showed that strong depolarisation could evoke ‘mode 2′ type behaviour in single cardiac L-type Ca²⁺ channels and this form of behaviour has also been shown to be enhanced by β-adrenergic stimulation (Ochi & Kawashima, 1990; Yue et al. 1990; Hirano et al. 1999). It is therefore possible that voltage-dependent potentiation of L-type Ca²⁺ channel currents (see Kamp & Hell, 2000 for review) results from the loss of voltage-dependent inactivation.