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

Abstract

L-type Ca²⁺ channel currents in native ventricular myocytes inactivate according to voltage- and Ca²⁺-dependent processes. This study sought to examine the effect of β-adrenergic stimulation on the contributions of voltage and Ca²⁺ to Ca²⁺ current decay. Ventricular myocytes were enzymatically isolated from guinea-pig hearts. Inward whole-cell Cd²⁺-sensitive L-type Ca²⁺ channel currents were recorded with the patch clamp technique and comparison was made between inward currents carried by Ca²⁺ and either Ba²⁺, Sr²⁺ or Na⁺. In control conditions the decay of Ca²⁺ currents was faster than Ba²⁺, Sr²⁺ or Na⁺ currents at negative voltages while at positive voltages there was no difference. The relationship between voltage and inactivation for Ca²⁺ currents was bell-shaped, while that for Ba²⁺, Sr²⁺, and Na⁺ currents was sigmoid. Thus depolarisation progressively replaced Ca²⁺-dependent inactivation in the fast phase of decay of Ca²⁺ channel currents with rapid voltage-dependent inactivation. In the presence of isoproterenol (isoprenaline) the decay of Ca²⁺ currents was faster than Ba²⁺, Sr²⁺ or Na⁺ currents at all measured voltages (-40 to +30 mV). The relationship between voltage and inactivation for Ca²⁺, Ba²⁺ and Sr²⁺ currents was bell-shaped, while that for Na⁺ currents was sigmoid with less inactivation than under control conditions. Therefore the fast phase of decay of Ca²⁺ channel currents was now almost entirely due to Ca²⁺. It is concluded that the relative contributions of Ca²⁺- and voltage-dependent mechanisms of inactivation of L-type Ca²⁺ channels in native cardiac myocytes are modulated by β-adrenergic stimulation influencing the amount of rapid voltage-dependent inactivation.

L-type Ca²⁺ channels in native cardiac myocytes inactivate according to two processes, membrane voltage- and Ca²⁺-induced inactivation. The relative contributions of these two processes to the decay of Ca²⁺ channel currents has been investigated for many years (see Pelzer et al. 1990 for review). The consensus of opinion holds that Ca²⁺ influx and Ca²⁺ release from the sarcoplasmic reticulum are largely responsible for the rapid phase of decay of Ca²⁺ channel currents (Bechem & Pott, 1985; Lee et al. 1985), the later slower phase of decay results from the voltage-dependent mechanism of inactivation. The bedrock of this consensus is the observation that currents carried by cations such as Ba²⁺, Sr²⁺ or Na⁺, which do not provoke ‘Ca²⁺-induced’ inactivation, decay more slowly than those carried by Ca²⁺ (Lee et al. 1985, but see Ferreira et al. 1997). Recently Mitarai et al. (2000) showed a strong voltage-dependent inactivation of Na⁺ current through Ca²⁺ channels in control conditions. This is in contrast to the classical representation of exceedingly slow decay of Ca²⁺ currents carried by Na⁺ (Matsuda, 1986). This novel observation calls into question previous evaluations of the contributions of voltage- and Ca²⁺-induced inactivation to the decay of Ca²⁺ channel currents (Imredy & Yue, 1994; Linz & Meyer, 1998; Puglishi et al. 1999; Sun et al. 2000). Mitarai et al. (2000) did not compare the decay of Ca²⁺ channel currents carried by Na⁺ with those carried by Ca²⁺. This study therefore set out to examine the decay of L-type Ca²⁺ channel currents carried by different cations in isolated ventricular myocytes of the guinea pig under both control conditions and following β-adrenergic stimulation. An examination of the literature surprisingly revealed that this is the first time that such an extensive comparison has been made in native cardiac myocytes.

In this study ryanodine was employed to block Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum. CICR significantly influences the rate of decay of L-type Ca²⁺ currents carried by Ca²⁺ in native myocytes (Mitchell et al. 1984; Callewaert et al. 1988; Wier et al. 1994; Adachi-Akahane et al. 1996; Sham, 1997; Puglisi et al. 1999). CICR is also enhanced during β-adrenergic stimulation (Hussein & Orchard, 1997; Song et al. 2001; Viatchenko-Karpinski & Gyorke, 2001). This variable component would be absent from the decay of Ca²⁺ channel currents carried by cations that did not induce CICR. The use of ryanodine excluded this source of variation and the experiments compared Ca²⁺ channel currents carried by different cations where only the influx of cations would influence inactivation.

The results show that the rapid phase of decay of Ca²⁺ channel currents recorded under control conditions in the native myocyte contains a large component of voltage-dependent inactivation. On the other hand, following β-adrenergic stimulation, the decay of Ca²⁺ channel currents is almost entirely due to Ca²⁺-induced inactivation. It is clear that the contribution of membrane voltage to the decay of L-type Ca²⁺ channels in cardiac myocytes has been underestimated.

METHODS

Cell preparation

All animal experiments were conducted according to the ethical standards of the Ministére Français de l'Agriculture (Licence number B37-261-4). Male guinea pigs (250-400 g) 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 that served as experimental chambers. The storage solution consisted of the standard extracellular solution described below. Dishes that contained myocytes were kept on the laboratory bench and used within 6-8 h of isolation.

Experimental procedures

Whole-cell current voltage clamp experiments 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. Data analysis was performed with Acquis1 and Origin 4.1 (Microcal Software, MA, USA). Once the whole-cell configuration of the patch clamp cell current recording technique (Hamill et al. 1981) had been achieved, isolated myocytes were voltage clamped at −80 mV. Voltage clamp protocols were delivered to the cells from this holding potential. The effect of changing extracellular divalent cations upon the apparent voltage dependence of inactivation caused by interfering with membrane surface charge was assessed with a double-pulse voltage clamp protocol to obtain the quasi-steady-state availability of Ca²⁺ channel currents carried by Ca²⁺, Ba²⁺, Sr²⁺ and Na⁺. The results from these experiments (not shown) revealed that exchanging Ba²⁺ for Ca²⁺ altered the voltage corresponding to half-inactivation (V_0.5) of Boltzmann relations fitted to the data (Origin 4.1) from −24 to −26 mV (n = 6), exchanging Sr²⁺ for Ca²⁺ altered the V_0.5 from −27 to −31 mV (n = 8), and exchanging Na⁺(EDTA) for Ca²⁺ altered V_0.5 from −25 to −35 mV (n = 8). In consequence, adjustments to the voltage clamp protocol took account of these membrane-screening effects. Comparisons of currents carried by Ca²⁺ and Ba²⁺ or Sr²⁺ were made from traces obtained at the same membrane voltages. Comparisons of currents carried by Ca²⁺ and Na⁺ were made between Na⁺ currents recorded 10 mV negative to those carried by Ca²⁺ in the same cells. Each voltage clamp protocol was preceded by a voltage step to −50 mV for Ca²⁺ and Ba²⁺ and −60 mV for Sr²⁺ and Na⁺(EDTA) 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). Cell currents were evoked by rectangular 1000 ms duration voltage clamp steps to between −50 and +80 mV for Ca²⁺ and Ba²⁺ and −60 mV and +70 mV for Sr²⁺ and Na⁺(EDTA) in 10 mV increments. 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 4-5 s. All experiments were conducted at room temperature (≈23 °C). Voltage-gated inwardly directed currents through L-type Ca²⁺ channels were defined as currents sensitive to 200 μM CdCl₂ (or 500 μM CdCl₂ in solution which contained 250 μM EDTA) Linz & Meyer (1998) discuss the limitations of this method. The cell current records obtained in the presence of Cd²⁺ were digitally subtracted from those recorded in the absence of Cd²⁺ using Acquis1.

Experimental solutions

The standard extracellular solution 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 adjusted to 7.4 with NaOH. Extracellular solutions used to superfuse cells during electrophysiological recording contained 100 mm NaCl, 40 mm TEACl, 5 mm CsCl, 10 μM TTX (citrate salt from Alomone Labs, Israel), 5 μM ryanodine (Sigma, France), 10 mm glucose, 10 mm Hepes, pH adjusted to 7.4 with NaOH. To this solution was added 2 mm of either CaCl₂, BaCl₂ or SrCl₂ or 250 μM EDTA-NaOH. The latter was used to chelate residual extracellular divalent cations and permit monovalent cation influx via Ca²⁺ channels (Lee & Tsien, 1984; Matsuda, 1986). It should be noted that none of the extracellular solutions contained added Mg²⁺ (Campbell et al. 1988). These solutions were used for experiments considered to correspond to control conditions. A second series of experiments were conducted to evaluate the effects of β-adrenergic stimulation, in these experiments the extracellular solutions also contained 100 nm isoproterenol. The ‘intracellular’ solution used to fill the patch pipettes contained (mm): 100 CsCl, 40 TEA-Cl, 5 EGTA-CsOH, 1.4 MgCl₂, 0.1 CaCl₂, 2 ATP-Mg²⁺, 0.1 GTP, 10 glucose, 10 Hepes, pH adjusted to 7.3 with CsOH. The estimated free concentrations of Mg²⁺ and Ca²⁺ in this solution were 1 mm and 1 nm, respectively.

RESULTS

Inactivation of I_Ca under control conditions

Figure 1 illustrates the decay of normalised cell currents obtained from three different representative ventricular myocytes. The currents in each myocyte were first recorded in the presence of extracellular Ca²⁺ and then in either the presence of extracellular Ba²⁺ (Fig. 1A), extracellular Sr²⁺ (Fig. 1B) or extracellular Na⁺-EDTA where the inward Ca²⁺ channel current is carried largely by extracellular Na⁺ (Fig. 1C). It is clear in each case that during a voltage step to −20 mV or its equivalent (-30 mV in Na⁺-EDTA) inactivation of the current carried by Ca²⁺ was faster than that carried by the other cation in the same cell. At 0 mV the difference between the currents carried by Ca²⁺ and the other cations was less marked, particularly for the initial rapid phase of inactivation. Voltage steps to +20 mV evoked cell currents whose fast initial phase of inactivation was the same when the Ca²⁺ current was carried by Ca²⁺ or Ba²⁺ (Fig. 1A), Ca²⁺ or Sr²⁺ (Fig. 1B) and Ca²⁺ or Na⁺ (Fig. 1C). This last result is surprising in the context that it is generally considered that currents carried by Ca²⁺ via the L-type Ca²⁺ channel in native cardiac myocytes inactivate more rapidly than those carried by either Ba²⁺, Sr²⁺ or Na⁺. These results (Fig. 1) suggest that the difference between the decay of currents carried by Ca²⁺ and another cation will depend upon the membrane voltage at which the currents are recorded.

Figure 1. Decay of L-type Ca²⁺ channel currents carried by different cations recorded under control conditions.

These records represent superimposed normalised cell currents obtained by 1000 ms duration voltage steps to −20 mV (left column), 0 mV (central column) and +20 mV (right column). A, B and C represent currents recorded from three different representative ventricular myocytes, where cell currents and superimposed records were obtained in Ca²⁺ and Ba²⁺ (A), Ca²⁺ and Sr²⁺ (B) and Ca²⁺ and Na⁺ (C). The cell currents carried by Na⁺ in C were recorded at a voltage 10 mV negative (i.e. −30, −10 and +10 mV) to that indicated in the figure (see Methods). The time scale in A is applicable to all pairs of records. The dotted lines indicate the 0 pA current level. The amplitude of cell currents recorded, respectively, at −20, 0 and +20 mV expressed in pA pF⁻¹ were as follows: Ca²⁺ (n = 36), −1.6 ± 0.1, −5.8 ± 0.3, −4.5 ± 0.2; Ba²⁺ (n = 13), −4.8 ± 0.9, −4.6 ± 0.6, −1.5 ± 0.1; Sr²⁺ (n = 11), −3.7 ± 0.2, −5.9 ± 0.3, −2.5 ± 0.1; Na⁺ (n = 12), −41 ± 3, −44 ± 2, −19 ± 1.

The effect of membrane voltage upon the decay of Ca²⁺ channels currents under control conditions is shown in Fig. 2. Inactivation of the Ca²⁺ channel current carried by Ca²⁺ (Fig. 2A) was bell-shaped with respect to membrane voltage with a maximum of inactivation at ≈0 mV. On the other hand the voltage dependence of inactivation of Ca²⁺ channel currents carried by Ba²⁺ (Fig. 2B), Sr²⁺ (Fig. 2C) and Na⁺ (Fig. 2D) were each approximately sigmoid which is what one would expect from an essentially voltage-dependent process.

Figure 2. The voltage dependence of the decay of L-type Ca²⁺ channel currents recorded under control conditions.

Cell currents were evoked by 1000 ms duration voltage steps to between −40 and +30 mV in 10 mV increments. The decay of inwardly directed currents through Ca²⁺ channels was quantified by measuring the amplitude of the current at fixed times following activation and expressing this relative to the initial peak amplitude of the current. Data are shown for the degree of inactivation recorded 10 ms (filled squares), 20 ms (filled circles), 50 ms (triangles), 100 ms (inverted triangles), 200 ms (diamonds), 500 ms (open squares) and 1000 ms (open circles) following activation. Symbols and bars represent means ±s.e.m. of data obtained from n myocytes. Three series of experiments were performed where currents were carried by Ca²⁺ (A) and either Ba²⁺ (B, n = 13), Sr²⁺ (C, n = 11) or Na⁺ (D, n = 12). Data for currents carried by Ca²⁺ were combined from the three series of experiments (n = 36).

The inactivation of currents carried by Ca²⁺ declined beyond the peak of the inactivation-voltage relationship. Since the inactivation of currents carried by other cations continued to increase, a point was reached where the two conditions coincided. At this point, voltage would be the major factor determining the inactivation of L-type Ca²⁺ channel currents. The following section shows how this conclusion is altered when experiments are conducted under conditions of β-adrenergic stimulation.

Inactivation of I_Ca following β-adrenergic stimulation

Figure 3 shows normalised cell currents recorded from three representative myocytes (A-C) exposed to 100 nm of the β-adrenergic agonist isoproterenol. In each cell at each of the illustrated voltages inactivation of the current carried by Ca²⁺ was more rapid and clearly distinct from that carried by Ba²⁺ (Fig. 3A), Sr²⁺ (Fig. 3B) or Na⁺ (Fig. 3C). Thus whereas under control conditions membrane depolarisation provoked the approach of inactivation of currents carried by Ca²⁺ and Ba²⁺, Sr²⁺ or Na⁺, the voltage dependence of inactivation following β-adrenergic stimulation appears to have been altered.

Figure 3. Decay of L-type Ca²⁺ channel currents carried by different cations recorded in the presence of isoproterenol.

Layout and description as for Fig. 1. A, B and C represent currents recorded from three different representative myocytes exposed to 100 nm isoproterenol. The time scale in A is applicable to all pairs of records. The amplitude of cell currents recorded, respectively, at −20, 0 and +20 mV expressed in pA pF⁻¹ were as follows: Ca²⁺ (n = 33), −21.4 ± 1.3, −28.9 ± 1.3, −16.6 ± 0.8; Ba²⁺ (n = 11), −42.2 ± 3.6, −20.6 ± 2.1, −5.6 ± 0.8; Sr²⁺ (n = 12), −37.2 ± 3.1, −25.2 ± 1.7, −9.4 ± 0.8; Na⁺ (n = 10), −139 ± 11, −73 ± 5, −20 ± 2.

The relationship between membrane voltage and inactivation of Ca²⁺ channel currents following β-adrenergic stimulation is shown in Fig. 4. As seen under control conditions (Fig. 2A) the relationship between inactivation of the currents carried by Ca²⁺ and membrane voltage was bell-shaped (Fig. 4A). This characteristic was enhanced by β-adrenergic stimulation and skewed towards more negative voltages such that the peak of inactivation was recorded at ≈-20 mV. In contrast to the results obtained under control conditions (Figs 2B and C) the relationship between inactivation of currents carried by Ba²⁺ and Sr²⁺ and membrane voltage was bell-shaped (Fig. 4B and C). Only the inactivation of currents carried by Na⁺ (Fig. 4D) had a sigmoid relationship with membrane voltage following β-adrenergic stimulation. This was reduced by comparison with control conditions (Fig. 2D).

Figure 4. The voltage dependence of the decay of L-type Ca²⁺ channel currents recorded in the presence of isoproterenol.

Layout, labels and analysis performed as described in Fig. 2 for cells here exposed to 100 nm isoproterenol. Symbols and bars represent means ± s.e.m. of data obtained from n myocytes. n = 33 for the data for Ca²⁺ (A) combined from the three series of experiments; n = 11 for Ba²⁺ (B); n = 12 for Sr²⁺ (C); n = 10 for Na⁺ (D).

These results indicate several radical changes in inactivation of whole-cell Ca²⁺ channel currents during β-adrenergic stimulation. This is clearly shown by the typical cell currents carried by Ba²⁺, Sr²⁺ and Na⁺ illustrated in Fig. 5. In control conditions (Fig. 5A-C), cell current decay recorded with voltage steps to between −20 and +20 mV accelerated with depolarisation. On the other hand in isoproterenol (Fig. 5D-F) the decay of currents carried by Ba²⁺ (Fig. 5D) and Sr²⁺ (Fig. 5E) sequentially slowed down with depolarisation. Only currents carried by Na⁺ (Fig. 5F) maintained voltage-dependent acceleration of decay in isoproterenol. The following section concentrates upon evaluating the effect of β-adrenergic stimulation upon inactivation of Ca²⁺ channel currents.

Figure 5. The effect of voltage upon the decay of L-type Ca²⁺ channel currents.

These traces represent normalised records obtained from 6 typical cells (A-F). Each group of traces was obtained from one myocyte and represents superimposed currents evoked by voltage steps to between −20 and +20 mV in 10 mV increments. Myocyte currents were recorded either under control conditions (A-C) or in the presence of 100 nm isoproterenol (D-F), in cells where currents were carried by Ba²⁺ (A and D), Sr²⁺ (B and E) or Na⁺ (C and F). The scale bar in D is common to all groups of records. The dotted lines represent the 0 pA current level. The values of peak currents recorded, respectively, at −20, −10, 0, +10 and +20 mV shown in this figure expressed in pA pF⁻¹ were as follows: A, −4.2, −6.3, −5.3, −3.4, −2.0; B, -3.6, −5.5, −5.1, −3.6, −2.2; C, −48, −47, −37, −32, −19; D, −45, −32, −20, −11, −6; E, −43, −39, −28, −18, −10; F, −152, −105, −69, −40, −18.

The effect of β-adrenergic stimulation upon inactivation of I_Ca

Figure 6 compares the decay of cell currents recorded under control conditions with those obtained following β-adrenergic stimulation. For the currents carried by divalent cations (Fig. 6A–C) it is clear that at −20 mV (Fig. 6, left column) isoproterenol is associated with a marked acceleration of inactivation. At 0 mV (Fig. 6, central column) this distinction is less obvious, with isoproterenol evoking more rapid inactivation of the current carried by Ca²⁺ (Fig. 6A) and slightly slower inactivation than control for the currents carried by Ba²⁺ (Fig. 6B) and Sr²⁺ (Fig. 6C). At +20 mV (Fig. 6, right column) the slower inactivation of currents carried by Ba²⁺ and Sr²⁺ following β-adrenergic stimulation is clear. These results therefore reveal an apparently complex effect of isoproterenol upon the inactivation of Ca²⁺ channel currents carried by divalent cations. In contrast, the effect of isoproterenol upon the inactivation of Ca²⁺ channel currents carried by Na⁺ is consistent: a slowing of inactivation compared with control conditions irrespective of the membrane voltage (Fig. 6D).

Figure 6. The effect of β-adrenergic stimulation upon the decay of L-type Ca²⁺ channel currents.

These graphs illustrate the effect of isoproterenol upon the decay of cell currents carried by Ca²⁺ (A), Ba²⁺ (B), Sr²⁺ (C) and Na⁺ (D). The time courses of the development of inactivation recorded either under control conditions (squares) or in the presence of 100 nm isoproterenol (circles) are compared for data recorded during voltage steps to −20 mV (left column), 0 mV (central column) and +20 mV (right column). The symbols and bars represent means ±s.e.m. for data obtained from n myocytes. n for control from Fig. 2 and for isoproterenol from Fig. 4.

The cause of the complexity of the effects of isoproterenol upon the inactivation of currents carried by divalent cations is shown in Fig. 7. Here data from the evaluation of the voltage dependence of inactivation (Fig. 2 and Fig. 4) have been extracted for two points in time; first, 20 ms after activation to follow the evolution of the initial rapid phase of decay of the currents (left column) and second, 200 ms after activation to evaluate the later slow phase of decay of the currents (right column). The effect of isoproterenol upon inactivation of currents carried by Ca²⁺ (Fig. 7A) represents an enhancement of the bell-shaped relationship with voltage seen under control conditions that is now also skewed towards negative voltages. The effect of isoproterenol upon inactivation of currents carried by Ba²⁺ (Fig. 7B) or Sr²⁺ (Fig. 7C) is clearly more complex. At neither 20 nor 200 ms following activation was the effect of isoproterenol either a simple addition to or enhancement of the situation recorded under control conditions. Thus at positive membrane potentials, inactivation was reduced. At negative membrane potentials, inactivation was enhanced. These results could be explained by a strong negative shift of the relationship between inactivation and membrane voltage, to which a form of supramaximal block of the process was added. However, the effects of isoproterenol upon the inactivation of Ca²⁺ channel currents carried by Na⁺ (Fig. 7D) clearly indicate a reduction of voltage-dependent inactivation, both for the fast and slow phases of decay.

Figure 7. The effect of β-adrenergic stimulation on the voltage dependence of decay of L-type Ca²⁺ channel currents.

Data corresponding to the relative amplitude of currents recorded 20 ms (left column) and 200 ms (right column) following activation were extracted from Figs 2 (squares: control) and 4 (circles: isoproterenol) to compare the effect of isoproterenol upon the voltage dependence of decay of currents carried by Ca²⁺ (A), Ba²⁺ (B), Sr²⁺ (C) and Na⁺ (D). Symbols and bars represent means ±s.e.m.n for control from Fig. 2 and for isoproterenol from Fig. 4.

The influence of Ca²⁺ upon inactivation of I_Ca

The experiments shown in Fig. 1 and Fig. 2 and 3 and 4 were conducted in matched pairs. For each myocyte, whether under control conditions (Fig. 1 and Fig. 2) or in the presence of 100 nm isoproterenol (Fig. 3 and Fig. 4), currents carried by Ca²⁺ and another cation were recorded. It is therefore possible to directly compare the two. For the purpose of the following calculation it was assumed that inactivation of currents carried by Ca²⁺ represents the total of inactivation, i.e. the sum of the processes of voltage- and Ca²⁺-induced inactivation. The inactivation of currents carried by the other cations was assumed, for the sake of the calculation, to represent inactivation proceeding without the process of Ca²⁺-induced inactivation. The difference between the inactivation of the currents carried by Ba²⁺, Sr²⁺ or Na⁺ and Ca²⁺ was then expressed as their proportion of total inactivation. This difference was taken, for the sake of discussion, to represent the proportion of inactivation due to Ca²⁺. The data in Fig. 8 analysed the initial rapid phase of inactivation recorded 20 ms following activation of the currents. The evaluation of the contribution of Ca²⁺ towards the process of inactivation was first examined by comparing the currents carried by Ca²⁺ and Ba²⁺ (Fig. 8A). Under control conditions there was a clear ‘voltage dependence’ of the contribution of Ca²⁺ to the total inactivation of the current. The fast phase of decay was entirely due to Ca²⁺ at −30 and −20 mV. At more positive voltages the proportion of the fast phase of decay due to Ca²⁺ was reduced, until at +20 and +30 mV little or none of the decay of the current could be assessed as being due to Ca²⁺. At these voltages therefore, the fast phase of decay of the Ca²⁺ current is entirely due to the process of voltage-dependent inactivation. Similar results were obtained when this data was evaluated from currents carried by Sr²⁺ (Fig. 8B) and Na⁺ (Fig. 8C) under control conditions. On the other hand, an entirely different picture emerges from the analysis of decay following β-adrenergic stimulation. When data was evaluated by comparing currents carried by Ca²⁺ and Ba²⁺ (Fig. 8A, right column) it was found that the fast phase of decay of the Ca²⁺ current was almost entirely dependent upon Ca²⁺ over the entire voltage range from −40 to +30 mV. Analysis of the fast phase of decay of Ca²⁺ and Sr²⁺ currents following β-adrenergic stimulation (Fig. 8B) was little different. The data obtained by comparing the fast phase of decay of currents carried by Ca²⁺ and Na⁺ following β-adrenergic stimulation (Fig. 8C) showed some reduction of the contribution of Ca²⁺ to inactivation with depolarisation. Nevertheless, compared with control conditions (Fig. 8C, left column) a large increase in the contribution of Ca²⁺ to the decay was observed with β-adrenergic stimulation (Fig. 8C, right column).

Figure 8. The contribution of Ca²⁺ to inactivation of L-type Ca²⁺ channel currents.

The difference between data comparing decay of Ca²⁺ and Ba²⁺ currents (A), Ca²⁺ and Sr²⁺ currents (B) and Ca²⁺ and Na⁺ currents (C) was calculated as the proportion of that recorded in Ca²⁺ under control conditions (left column) and in the presence of 100 nm isoproterenol (right column). See text for details.

DISCUSSION

Two principal points arise from this investigation. Firstly, under clearly defined control conditions, the decay of L-type Ca²⁺ channel currents carried by Ca²⁺, Ba²⁺, Sr²⁺ and Na⁺ can be the same. Secondly, when comparing the relationship between membrane voltage and decay of L-type Ca²⁺ channel currents carried by the divalent cations Ba²⁺ and Sr²⁺, there are marked differences in their character when recorded under control conditions where the relationship appeared to be voltage dependent, and following β-adrenergic stimulation where the relation appeared to be ion dependent. This is the first time that these characteristics of native L-type Ca²⁺ channels have been clearly demonstrated.

The first observation contradicts the tenet that decay of L-type Ca²⁺ channel currents is dominated by Ca²⁺-induced inactivation (Eckert & Chad, 1984; Kass & Sanguinetti, 1984; Mentrard et al. 1984; McDonald et al. 1994; see Anderson, 2001 for recent review). This study has clearly defined the conditions under which the comparison of the decay of L-type Ca²⁺ currents carried by different cations was made. In control conditions the Ca²⁺ current was neither stimulated with β-adrenergic agonists nor enhanced by protein kinase A-activating agents. In all experiments CICR was blocked by ryanodine and the recording pipette contained EGTA. Mitarai et al. (2000) emphasised the need to clearly separate ‘control’ from ‘agonist’ conditions for the evaluation of the contribution of voltage-dependent inactivation to the decay of cardiac Ca²⁺ currents. Prior studies have not necessarily made this distinction (Linz & Meyer, 1998; Puglisi et al. 1999; Sun et al. 2000) and, in fact, a number of evaluations of the contribution of membrane voltage to inactivation of cardiac L-type Ca²⁺ currents were made under conditions that corresponded to those of β-adrenergic stimulation (Fukushima & Hagiwara, 1985; Matsuda, 1986; Linz & Meyer, 1998). The reasons for this are various, though enhancement and/or maximisation of the Ca²⁺ current was frequently advanced since it was not appreciated that such treatment would fundamentally alter the character of voltage-dependent inactivation (Mitarai et al. 2000). The contribution of Ca²⁺ to total inactivation, which itself was recorded under control conditions in these studies, was therefore seriously over-estimated. This study clearly shows that voltage-dependent inactivation plays a large part in the decay of native L-type Ca²⁺ channels under control conditions. It is emphasised that it is not suggested that inactivation of native L-type Ca²⁺ channels is either voltage-dependent or Ca²⁺-dependent. Rather it is suggested that the two processes contribute according to the conditions. Thus when the rapid decay of currents carried by Ca²⁺ and Ba²⁺, Sr²⁺ or Na⁺ coincide (Fig. 1) the later and slower decay results principally from Ca²⁺-induced inactivation. In this manner the decay of currents carried by Ca²⁺ under control conditions is finally more extensive than for currents carried by other cations.

The second point concerns the change in the character of inactivation of Ca²⁺ channel currents carried by Ba²⁺ and Sr²⁺ caused by β-adrenergic stimulation. In control conditions their sigmoid relationship between inactivation and membrane voltage supports the major role for membrane voltage in determining their rate of decay (Kass & Sanguinetti, 1984; Mentrard et al. 1984; Lee et al. 1985; Hadley & Hume, 1987). Their bell-shaped relationship between inactivation and voltage following β-adrenergic stimulation could suggest an effect of Ba²⁺ and Sr²⁺ upon the process of ‘Ca²⁺-dependent’ inactivation. However, U- or bell-shaped relationships between inactivation and membrane voltage can result from phenomena that do not involve ion-dependent inactivation (Gera & Byerly, 1999; see Stotz & Zamponi, 2001 for recent review). In this study, under the same conditions and in the same cells, the relationship between inactivation and voltage for currents carried by Ca²⁺ was clearly bell-shaped. But in isoproterenol, the relationship between inactivation of currents carried by Na⁺ and membrane voltage was not bell-shaped. This indicates that β-adrenergic stimulation had not fundamentally altered the form of the relationship between membrane voltage and inactivation that could otherwise have explained the results obtained with Ba²⁺ and Sr²⁺. It is therefore suggested that in isoproterenol the currents carried by Ba²⁺ and Sr²⁺ show ‘ion-dependent’ inactivation (Ferreira et al. 1997). Further studies are required to determine whether this is simply a consequence of increased influx and thus higher intracellular concentrations of these divalent cations which would stimulate the process of Ca²⁺-induced inactivation, or whether the sensitivity of the process to divalent cations had been enhanced by β-adrenergic stimulation.

A question which arises from this study is how can one evaluate the respective roles of membrane voltage and Ca²⁺/ion-dependent inactivation in the decay of L-type Ca²⁺ channel currents? In control conditions adequate comparisons could be made for currents carried by Ca²⁺ and Ba²⁺, Ca²⁺ and Sr²⁺, and Ca²⁺ and Na⁺ when membrane charge-screening had been compensated. It is clear that with depolarisation the influence of Ca²⁺ influx declines. Where this study differs from previous evaluations is that this decline is shown to be due to the voltage-dependent development of rapid inactivation which occurs irrespective of whether currents are carried by Ba²⁺, Sr²⁺ or Na⁺. In isoproterenol, comparisons between Ca²⁺ and Ba²⁺, and Ca²⁺ and Sr²⁺, suggest a voltage independence and virtual total dependence of the decay of L-type Ca²⁺ channel currents upon Ca²⁺-induced inactivation. Similar conclusions were reached by Bechem & Pott (1985) and Imredy & Yue (1994) who evaluated the contribution of Ca²⁺ to total inactivation under experimental conditions which also corresponded to those of β-adrenergic stimulation. However, since Ba²⁺ and Sr²⁺ currents might share the process of ‘Ca²⁺-induced’ inactivation, the direct comparison with Ca²⁺ currents would not clearly separate the mechanisms of inactivation. Therefore, the comparison of currents carried by Ca²⁺ and Na⁺ seems to most adequately represent, respectively, total inactivation and inactivation associated with membrane voltage, once membrane surface charge screening has been accounted for. The contribution of Ca²⁺ to total inactivation in isoproterenol is then shown to be large and to decline only slightly with depolarisation. These data suggest an overall dominance of Ca²⁺-induced inactivation for the L-type Ca²⁺ current following β-adrenergic stimulation.

The physiological interest of this study lies in the increased emphasis upon the process of voltage-dependent inactivation under basal conditions compared with previous evaluations of the relative contributions of Ca²⁺ and voltage-dependent inactivation to the decay of L-type Ca²⁺ channel currents in native cardiac myocytes. It is also clear that their relative importance can be modulated under the influence of sympathetic stimulation. Thus failure of, or interference with, the process of Ca²⁺-induced inactivation might actually have little effect under control conditions but dramatically influence the Ca²⁺ current during β-adrenergic stimulation.