Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes

Abstract

The effects of changing cytosolic [Mg²⁺] ([Mg²⁺]_i) on l-type Ca²⁺ currents were investigated in rat cardiac ventricular myocytes voltage-clamped with patch pipettes containing salt solutions with defined [Mg²⁺] and [Ca²⁺]. To control [Mg²⁺]_i and cytosolic [Ca²⁺] ([Ca²⁺]_i), the pipette solution included 30 mm citrate and 10 mm ATP along with 5 mm EGTA (slow Ca²⁺ buffer) or 15 mm EGTA plus 5 mm BAPTA (fast Ca²⁺ buffer). With pipette [Ca²⁺] ([Ca²⁺]_p) set at 100 nm using a slow Ca²⁺ buffer and pipette [Mg²⁺] ([Mg²⁺]_p) set at 0.2 mm, peak l-type Ca²⁺ current density (I_Ca) was 17.0 ± 2.2 pA pF⁻¹. Under the same conditions, but with [Mg²⁺]_p set to 1.8 mm, I_Ca was 5.6 ± 1.0 pA pF⁻¹, a 64 ± 2.8% decrease in amplitude. This decrease in I_Ca was accompanied by an acceleration and a –8 mV shift in the voltage dependence of current inactivation. The [Mg²⁺]_p-dependent decrease in I_Ca was not significantly different when myocytes were preincubated with 10 μm forskolin and 300 μm 3-isobutyl-1-methylxanthine and voltage-clamped with pipettes containing 50 μm okadaic acid, to maximize Ca²⁺ channel phosphorylation. However, when myocytes were voltage-clamped with pipettes containing protein phosphatase 2A, to promote channel dephosphorylation, I_Ca decreased only 25 ± 3.4% on changing [Mg²⁺]_p from 0.2 to 1.8 mm. In the presence of 0.2 mm[Mg²⁺]_p, changing channel phosphorylation conditions altered I_Ca over a 4-fold range; however, with 1.8 mm[Mg²⁺]_p, these same manoeuvres had a much smaller effect on I_Ca. These data suggest that [Mg²⁺]_i can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca²⁺]_p to 1, 100 or 300 nm also showed that the [Mg²⁺]_p-induced reduction of I_Ca was smaller at the lowest [Ca²⁺]_p, irrespective of channel phosphorylation conditions. This interaction between [Ca²⁺]_i and [Mg²⁺]_i to modulate I_Ca was not significantly affected by ryanodine, fast Ca²⁺ buffers or inhibitors of calmodulin, calmodulin-dependent kinase and calcineurin. Thus, physiologically relevant [Mg²⁺]_i modulates I_Ca by counteracting the effects of Ca²⁺ channel phosphorylation and by an unknown [Ca²⁺]_i-dependent mechanism. The magnitude of these effects suggests that changes in [Mg²⁺]_i could be critical in regulating l-type channel gating.

Cytosolic [Mg²⁺] ([Mg²⁺]_i) in cardiac myocytes appears to be 0.6–1.3 mm (Buri & McGuigan, 1990; Hongo et al. 1994; Watanabe & Konishi, 2001) and is largely buffered in the cytosol by a variety of diffusible molecules (e.g. ATP) and proteins (Robertson et al. 1981; Fabiato, 1983; Konishi & Berlin, 1993). Changes in [Mg²⁺]_i can have marked effects on fluxes through ion channels in cardiac myocytes (Agus et al. 1989; White & Hartzell, 1989; Agus & Agus, 2001). The first study of [Mg²⁺]_i effects on l-type Ca²⁺ current (I_Ca) in frog myocytes showed that, under appropriate conditions (see below), increasing [Mg²⁺]_i between 0.3 and 3.0 mm decreased I_Ca more than 50% (White & Hartzell, 1988). More recent studies in frog and guinea-pig myocytes have confirmed the marked inhibitory actions of increased [Mg²⁺]_i on I_Ca (Yamaoka & Seyama, 1996a,b, 1998; Pelzer et al. 2001; Yamaoka et al. 2002); however, none have shown the large changes of current around physiologically relevant [Mg²⁺]_i reported by White & Hartzell (1988). These recent results therefore raise the issue of whether [Mg²⁺]_i is a physiologically important regulator of Ca²⁺ channel function.

Two general mechanisms could explain how Mg²⁺ regulates Ca²⁺ fluxes through l-type channels: alteration of ion permeation and modulation of channel gating properties. Cytosolic Mg²⁺ concentrations up to 10 mm do not decrease divalent cation conductance through single l-type Ca²⁺ channels (Kuo & Hess, 1993; Yamaoka & Seyama, 1998), so that it is unlikely that the reported effects of cytosolic Mg²⁺ on macroscopic I_Ca (White & Hartzell, 1988; Agus et al. 1989; Yamaoka & Seyama, 1996a,b, 1998; Pelzer et al. 2001) result from block of Ca²⁺ permeation through the channel pore. For this reason, we have focused on mechanisms by which [Mg²⁺]_i could alter l-type channel gating properties.

l-type Ca²⁺ channel gating is regulated by at least three factors: membrane potential (V_m), cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and channel phosphorylation (McDonald et al. 1994). In this regard, a 10-fold increase in [Mg²⁺]_i has been shown to produce a small negative shift in the V_m dependence for inactivation of Cd²⁺-sensitive Ba²⁺ current (Hartzell & White, 1989). Channel phosphorylation state also appears to be important in [Mg²⁺]_i-dependent regulation of I_Ca. Earlier studies showed that increased [Mg²⁺]_i inhibited I_Ca most prominently under conditions of high channel phosphorylation (White & Hartzell, 1988; Agus et al. 1989). Under basal, presumably low phosphorylation conditions, inhibitory actions of [Mg²⁺]_i were less marked or not observed. Recent studies (Yamaoka & Seyama, 1998; Pelzer et al. 2001) suggest that this less pronounced reduction of I_Ca under basal conditions might reflect a shift in inhibitory [Mg²⁺] from a micromolar to millimolar range when the Ca²⁺ channel is phosphorylated and/or an inability of Mg²⁺ to modulate unphosphorylated channels. Finally, only one study examined how [Mg²⁺]_i influences Ca²⁺-dependent regulation of Ca²⁺ channels (Yamaoka & Seyama, 1996a), and depending on [Mg²⁺]_i, changes in [Ca²⁺]_i increased or decreased I_Ca. The apparent complexity of these Mg²⁺ actions on I_Ca suggests that the mechanisms by which [Mg²⁺]_i modulates l-type channel gating warrant further study.

The purpose of this study was therefore to determine whether physiologically relevant concentrations of cytosolic Mg²⁺ affect mechanisms, e.g. V_m, Ca²⁺, and channel phosphorylation, that regulate l-type channel gating. A whole-cell patch-clamp technique was used to measure l-type I_Ca density while dialysing cells with a pipette solution containing 40 mm Mg²⁺ buffers (30 mm citric acid and 10 mm ATP) to rapidly control [Mg²⁺]_i levels. We found that increasing pipette [Mg²⁺] from 0.2 mm to 1.8 mm suppressed I_Ca density by ∼70%. This inhibitory effect was enhanced by [Ca²⁺]_i but inhibited by channel dephosphorylation. However, neither V_m-dependent nor Ca²⁺/calmodulin-dependent mechanisms appeared to account for the marked reduction of I_Ca density by [Mg²⁺]_i.

Portions of this work have appeared previously as a preliminary communication (Wang & Berlin, 2002).

Methods

Cell isolation

Adult rat ventricular myocytes were isolated enzymatically as previously described (Mitra & Morad, 1985) from male Sprague–Dawley rats (200–225 g). Animals received an intraperitoneal injection of sodium pentobarbitone (50–100 mg kg⁻¹), and after full anaesthesia was achieved, a thoracotomy was performed to rapidly remove the heart, in accordance with the procedures approved by the Institutional Animal Care and Use Committee of the University of Medicine and Dentistry of New Jersey. Following isolation, myocytes were stored in a refrigerator and used within 1–8 h.

Measurement of membrane currents

Myocytes were placed in a chamber mounted on an inverted microscope (Nikon Inc., Japan) and superfused with a modified Tyrode solution (see Solutions below). Ca²⁺ current was measured in the whole-cell configuration with patch pipettes having resistances of 1.0–1.5 MΩ when filled with pipette solutions (see below). Outward K⁺ current was blocked by Cs⁺ and tetraethylammonium ions (TEA) in the pipette solution, while Na⁺ current was suppressed by addition of 30 μm tetrodotoxin (TTX) to the Tyrode solution and by depolarizing V_m to –40 mV with ramp pulses prior to test protocols (Nilius et al. 1985). All experiments were performed at room temperature. Experiments were conducted when whole-cell voltage clamps had time constants ranging from 100 to 300 μs without series resistance or capacitance compensation. Cell capacitance was estimated by integrating current elicited by 5 mV depolarizations from the holding potential of –70 mV

Experimental protocols

Cells were depolarized every 30 s from a holding potential of –70 mV to –40 mV with a 1 s ramp and then depolarized to a test potential of 0 mV for 200 ms. In some experiments, the I–V relationships were also obtained periodically by varying the test potential between –30 and +60 mV (in 10 mV increments) at 0.2 Hz. Displayed current records were obtained after 5 min in the whole-cell configuration to allow adequate intracellular dialysis (see Results), unless otherwise indicated. Data were analysed using pCLAMP software, version 8.0 (Axon Instruments, Union City, CA, USA), and I_Ca was calculated as 200 μm CdCl₂-sensitive difference current. Displayed membrane currents are current recordings shown without linear leak subtraction, unless otherwise indicated.

In experiments that monitored indo-1 loading, fluorescence intensity at 410 nm (the isosbestic point of this indicator in our system) was measured during illumination with 360 nm light using methodologies previously described in Konishi & Berlin (1993).

Solutions

The pipette solution was composed of (mm): 100 caesium gluconate, 10 Pipes (caesium salt), 15 TEACl, 0.5 NaH₂PO₄, 0.1 Tris-GTP, 5 EGTA along with ATP, Mg-ATP, citric acid, magnesium citrate, MgCl₂ and CaCl₂ to produce free [Mg²⁺] of 0.2, 0.6 and 1.8 mm, pH 7.2, at specified free [Ca²⁺] of 1, 100 and 300 nm. Free [Mg²⁺] and [Ca²⁺] were calculated using a computer program (WinMAXC 2.40 obtained at http://stanford.edu/~cpatton/maxc.html). A second series of experiments was carried out using the same pipette solution with 5 mm BAPTA and 15 mm EGTA. In some experiments, indo-1 (K⁺ salt) was added to the pipette solution at a final concentration of 100 μm. The superfusion solution was a modified Tyrode solution containing (mm): 145 NaCl, 4 KCl, 2 CaCl₂, 10 Hepes, 1 MgCl₂, and 10 glucose, pH 7.4. When noted, CaCl₂ in this solution was decreased to 0.5 mm.

Reagents

Unless specified, reagents were obtained from Sigma Chemical Corp. (St Louis, MO, USA). Autoinhibitory peptide (AIP; BioMol, Plymouth Meeting, PA, USA), calcineurin autoinhibitory peptide (CAP; CalBiochem, San Diego, CA, USA), okadaic acid (OA; CalBiochem), protein phosphatase 2A (PP_2A; Upstate Biochemicals Inc., Lake Placid, NY, USA), and the potassium salt of indo-1 (Molecular Probes, Eugene, OR, USA) were added directly to pipette solutions. Several reagents purchased from CalBiochem (cyclosporine A, 3-isobutyl-1-methylxanthine (IBMX), KN-93, ryanodine, TTX and W₇) and forskolin (BioMol) were prepared as concentrated stock solutions that were applied to bathing solutions 30 min prior to experiments. When DMSO or MeOH was used as the solvent for stock solutions, the final concentration in experimental solutions was less than or equal to 0.1%, and blank solutions containing 0.1% DMSO or MeOH were also prepared for control experiments.

Data analysis

Data are expressed as means ±s.e.m. for the number of cells indicated. Significance was determined using anova and Student's t test in commercial software (SigmaPlot, SPSS Inc., Chicago, IL, USA, and JMP IN, Duxbury, Pacific Grove, CA, USA). A P value less than 0.05 was considered statistically significant. The percentage change, confidence intervals (95%) and s.e.m. for current density ratios were calculated using Fieller's Theorem (Goldstein, 1964).

Results

Effect of [Mg²⁺]_i on calcium current

To assess the effects of [Mg²⁺]_i on whole-cell I_Ca, time diaries of I_Ca were recorded for test depolarizations to 0 mV, elicited every 30 s, when cells were voltage-clamped with patch electrodes containing different concentrations of Mg²⁺ ([Mg²⁺]_p) in the presence of 100 nm pipette [Ca²⁺] ([Ca²⁺]_p). In the myocytes dialysed with 0.2 mm[Mg²⁺]_p, I_Ca increased for 1–3 min after patch break-through followed by a long period of rundown before the current finally stabilized approximately 20 min into a 30 min observation period (Fig. 1A). The initial increase of I_Ca was probably caused by the relief from Mg²⁺ block of I_Ca due to the reduction of [Mg²⁺]_i from a resting level of [Mg²⁺]_i (∼1 mm) to 0.2 mm. The secondary rundown is thought to result from the washout of important cytoplasmic constituents (McDonald et al. 1994). In the myocytes dialysed with 1.8 mm[Mg²⁺]_p, I_Ca at 30 s after patch break-through was much smaller and, with time, declined faster and to a much lower level than that with 0.2 mm[Mg²⁺]_p. The time diaries show that I_Ca with 1.8 mm[Mg²⁺]_p was 60%, 47%, 37%, 35%, 32%, 31% and 22% of I_Ca with 0.2 mm[Mg²⁺]_p at 30 s, 1 min, 3 min, 5 min, 10 min, 15 min and 30 min after patch break-through, respectively. Representative currents at each of these time points are shown in Fig. 1B. Thus, differences in current density changed rapidly during the first 3–5 min after patch break-through and thereafter changed slowly.

Figure 1. L-type calcium current (I_Ca) recorded in rat ventricular myocytes dialysed with low and high [Mg²⁺]_p.

A, time diaries of I_Ca in rat ventricular myocytes depolarized to 0 mV from a holding potential of –40 mV during dialysis with pipette solutions containing 0.2 mm and 1.8 mm[Mg²⁺]_p. Time 0 coincides with patch break-through. Data are means with s.e.m. displayed for 5, 10, 15, 20, 25 and 30 min time points. The numbers of experiments are indicated in parentheses. Letters (a–f) in each time course correspond to the displayed currents. Inset: time course of indo-1 loading in patch-clamped myocytes. Fluorescence intensity was normalized to the maximal level measured in each of 5 cells (shown with different symbols). The continuous curve is the best exponential function with a time constant of 238 s. B, superimposed sample currents, continuous and dashed tracings were recorded with 0.2 mm and 1.8 mm[Mg²⁺]_p, respectively.

The period of rapid change in relative current densities in cells voltage-clamped with 0.2 and 1.8 mm[Mg²⁺]_p might be an indicator of dialysis from pipette solution to the cytosol. To test this assertion, the time course of indo-1 loading was examined in five cells that, when voltage-clamped, displayed an uncompensated time constant for decay of current during 5 mV depolarizations of 0.3 ms. Indo-1 loading was monitored by measuring fluorescence intensity at the isosbestic point for this Ca²⁺ indicator. These experiments showed that the time constant for increasing fluorescence intensity was 4 min (Fig. 1A, inset). This finding is consistent with our previous work (Berlin & Konishi, 1993). Given these data, in subsequent experiments, we waited for 5 min after patch break-through before I_Ca was measured. Within 5 min of establishing a whole-cell patch-clamp, cellular loading of small molecules, such as a fluorescent Ca²⁺ indicator, would be greater than 70% complete. Given the 40 mmol l⁻¹ of Mg²⁺ buffers in our patch electrode solutions, this would imply that approximately 25 mmol l⁻¹ of exogenous Mg²⁺ buffers, i.e. 20 mm citrate along with 70% of the difference between cytosolic and patch ATP concentrations, would have diffused into the cytosol within this time period. Such a large exogenous buffer concentration should be sufficient to overwhelm endogenous cytosolic Mg²⁺ buffering capacity and thereby gain control of [Mg²⁺]_i.

The V_m dependence of I_Ca was determined by a series of voltage pulses from –30 to +60 mV, as described in Methods. [Mg²⁺]_p effects on the I–V relationship for I_Ca are illustrated in Fig. 2A. When [Mg²⁺]_p was increased from 0.2 mm to 0.6 mm (not shown) and 1.8 mm, peak I_Ca amplitude was decreased by 56 ± 3.7%(n= 5) and 68 ± 3.5% (n= 5), respectively. Accounting for all experiments, including those where I–V relationships were not measured, increasing [Mg²⁺]_p from 0.2 to 1.8 mm decreased peak I_Ca measured at 0 mV by 64 ± 2.8% (n= 10). Increasing [Mg²⁺]_p also shifted the peak of the I–V relationship 5–10 mV in the negative direction and accelerated the rate of current inactivation, as shown by the normalization of current amplitudes in Fig. 2B.

Figure 2. Effect of [Mg²⁺]_p on I_Ca.

A, current–voltage relationship of I_Ca in myocytes dialysed with 0.2 mm and 1.8 mm[Mg²⁺]_p. Currents were recorded 5 min after establishing the whole-cell patch-clamp configuration. Data are means and s.e.m., with the number of experiments indicated in parentheses. B, average I_Ca tracings recorded at a test potential of 0 mV in rat ventricular myocytes dialysed with (1) 0.2 mm[Mg²⁺]_p, (2) 1.8 mm[Mg²⁺]_p and (3) 1.8 mm[Mg²⁺]_p, normalized relative to that with 0.2 mm[Mg²⁺]_p.

Effect of Mg²⁺ on the V_m dependence of calcium current

The observation that a decrease in I_Ca, when increasing [Mg²⁺]_p, is accompanied by a shift of the I–V relationship towards negative V_m could be interpreted as an effect of [Mg²⁺]_p on the V_m dependence of Ca²⁺ channel gating. Furthermore, the decrease in I_Ca that accompanies this leftward shift in the I–V relationship could imply that the V_m dependence of channel inactivation is also shifted to more negative potentials. To examine this possibility, the V_m dependence of channel inactivation was estimated with a two-pulse protocol consisting of a 400 ms prepulse (from –90 to +60 mV in 10 mV increments) followed, after a 3 ms interval at –40 mV, by a 200 ms test depolarization to 0 mV. Figure 3 shows the resulting inactivation curves at 0.2 mm and 1.8 mm[Mg²⁺]_p. With 0.2 mm[Mg²⁺]_p, the curve exhibited a characteristic ‘U’-shape, i.e. inactivation reached a maximum with prepulses to 0 mV but decreased with prepulses positive of 0 mV. However, at the higher [Mg²⁺]_p, the degree of current inactivation was more complete at positive V_m. Since V_m-dependent inactivation of l-type channels requires a long period to reach a pseudo-steady state (Hadley & Lederer, 1991), this protocol was repeated using 3 s prepulses. Results similar to those with 400 ms prepulses were observed. With both protocols, leftward shifts in the inactivation curves were observed. With 400 ms prepulses, increasing [Mg²⁺]_p from 0.2 to 1.8 mm shifted the V_m for half-maximal inactivation by −8.1 ± 0.7 mV (n= 5). The shift was −7.7 ± 1.0 mV (n= 5) with 3 s prepulses. These results suggested that [Mg²⁺]_p can affect the V_m-dependence of channel gating. It should be pointed out that the degree of this shift is small enough that, at –40 mV, the change in Ca²⁺ channel availability is minor, and therefore, a shift in V_m-dependent inactivation is unlikely to account for the marked decrease of I_Ca observed with higher [Mg²⁺]_p.

Figure 3. Effect of [Mg²⁺]_p on inactivation curves for I_Ca.

The V_m dependence of channel inactivation was measured at 5 min after break-through into the whole-cell patch-clamp configuration with a 2-pulse protocol. I_Ca at 0 mV after a given prepulse (I_Ca(T)) is divided by I_Ca at 0 mV after a prepulse to –60 mV (I_Ca(C)). Data are means and s.e.m., with the number of experiments indicated in parentheses for each [Mg²⁺]_p.

Ca²⁺ channel phosphorylation and Mg²⁺ effects on Ca²⁺ current

l-type Ca²⁺ channels are known to be regulated by channel phosphorylation (McDonald et al. 1994) and the phosphorylation state of the channel has been reported to be important in determining Mg²⁺ effects on I_Ca (White & Hartzell, 1988; Agus et al. 1989; Yamaoka & Seyama, 1998; Pelzer et al. 2001; Yamaoka et al. 2002). To investigate how the modulation of l-type Ca²⁺ channels by [Mg²⁺]_p is affected by channel phosphorylation, we conducted a series of experiments measuring currents where l-type Ca²⁺ channels were likely to be in phosphorylated and dephosphorylated states, at low (0.2 mm) and high (1.8 mm) [Mg²⁺]_p. To increase channel phosphorylation, cardiac myocytes were first preincubated for 30 min with 10 μm forskolin to activate adenylate cyclase and 300 μm 3-isobutyl-1-methylxanthine (IBMX) to inhibit cAMP and cGMP phosphodiesterases. l-type Ca²⁺ current was then measured 5 min after patch break-through with pipette solutions containing 50 μm okadaic acid (OA) to inhibit protein phosphatases that could dephosphorylate Ca²⁺ channels. This manoeuvre has been found to increase the I_Ca in frog and guinea-pig myocytes (Yamaoka & Seyama, 1998; Pelzer et al. 2001), presumably by cAMP-mediated phosphorylation of l-type Ca²⁺ channels.

Our experiments showed that in the presence of forskolin, IBMX and OA, I_Ca was dramatically increased to densities at 0 mV of 30–40 pA pF⁻¹ with 0.2 mm[Mg²⁺]_p (36.3 ± 2.1 pA pF⁻¹, n= 3). To minimize voltage errors due to this high current density, extracellular Ca²⁺ concentration ([Ca²⁺]_o) was therefore reduced from 2.0 to 0.5 mm. With 0.5 mm[Ca²⁺]_o, I_Ca at 0 mV was 16.8 ± 1.4 pA pF⁻¹ (n= 5) with 0.2 mm[Mg²⁺]_p in cells exposed to forskolin, IBMX and OA. This current density was not significantly different from that observed under basal conditions with 2.0 mm[Ca²⁺]_o.

To evaluate the effect of [Mg²⁺]_p on I_Ca, we first repeated experiments in Fig. 2A with 0.5 mm[Ca²⁺]_o (Fig. 4A and B, left). Under these conditions, increasing [Mg²⁺]_p from 0.2 mm to 1.8 mm decreased peak I_Ca amplitude by 75 ± 2.4% (n= 6) and shifted the peak of the I–V relationship 5–10 mV in the negative direction (Fig. 4A, continuous curves). This reduction of I_Ca was not statistically different from that observed with 2.0 mm[Ca²⁺]_o, so changing [Ca²⁺]_o appeared to have no effect on the decrease of I_Ca induced by increasing [Mg²⁺]_p. In the presence of forskolin, IBMX and OA, peak I_Ca amplitude with 1.8 mm[Mg²⁺]_p was 3.3 ± 0.4 pA pF⁻¹ (n= 7). Thus, under these conditions, increasing [Mg²⁺]_p from 0.2 mm to 1.8 mm produced a 79 ± 1.7% inhibition on peak I_Ca amplitude (Fig. 4A, dashed curves and Fig. 4B, right). Additionally, higher [Mg²⁺]_p caused a –5 mV shift in the peak of the I–V relationship. Thus, increasing [Mg²⁺]_i markedly reduced I_Ca under conditions promoting l-type Ca²⁺ channel phosphorylation.

Figure 4. Effect of [Mg²⁺]_p on I_Ca in high phosphorylation conditions.

A, current–voltage relationships for I_Ca in myocytes dialysed with 0.2 mm and 1.8 mm[Mg²⁺]_p in control (continuous curves) and high phosphorylation conditions (dashed curves) in the absence and presence of 10 μm forskolin (FSK), 300 μm IBMX and 50 μm OA, respectively, when [Ca²⁺]_o was set at 0.5 mm. Currents were recorded at 5 min after break-through into the whole-cell patch-clamp configuration. Data are means and s.e.m., with the number of experiments indicated in parentheses. B, tracings of typical I_Ca records at a test potential of 0 mV in rat ventricular myocytes dialysed with 0.2 mm (•) and 1.8 mm[Mg²⁺]_p (▴) in control conditions, and 0.2 mm (○) and 1.8 mm[Mg²⁺]_p (▵) in high phosphorylation conditions.

To induce channel dephosphorylation, the catalytic subunit of protein phosphatase 2A (PP_2A; 5 units ml⁻¹) was included in the pipette solution. PP_2A was chosen because l-type channels are complexed with stoichiometric amounts of PP_2A (Davare et al. 2000) and PP_2A is present in the heart (Herzig & Neumann, 2000). Furthermore, PP_2A activity has no requirement for Mg²⁺ and Ca²⁺ (Herzig & Neumann, 2000; Rusnak & Mertz, 2000) so that this enzyme should be insensitive to the experimentally induced changes in cytosolic concentrations of these divalent cations.

The extent of cell dialysis with a protein, such as PP_2A, is unknown in our experiments. For this reason, the time course of I_Ca was compared in the presence and absence of PP_2A (Fig. 5A and Fig. 1, respectively). In cells superfused with 2 mm Ca²⁺-containing Tyrode solution and voltage-clamped with patch pipette solutions containing 0.2 mm[Mg²⁺]_p and PP_2A, the initial rate of current rundown was more rapid than in the absence of PP_2A. After 5 min, average I_Ca density at 0 mV was 9.8 ± 1.3 pA pF⁻¹ (n= 12), a level 63 ± 3.7% of that measured in the absence of PP_2A (Fig. 5A). This decrease in I_Ca is statistically significant and is consistent with the results of duBell et al. (1996). Representative currents are shown in Fig. 5A, inset. Control experiments also showed that adding the enzyme carrier solution to the 0.2 mm Mg²⁺-containing pipette solution had no effect on I_Ca amplitude or kinetics. Thus, a 5-min period of cell dialysis appears to be sufficient for PP_2A to produce significant effects on I_Ca.

Figure 5. Effect of [Mg²⁺]_p on I_Ca in low phosphorylation conditions.

A, time diaries of I_Ca in rat ventricular myocytes depolarized to 0 mV from a holding potential of –40 mV during dialysis with 0.2 mm and 1.8 mm[Mg²⁺]_p electrode solution containing 5 units ml⁻¹ PP_2A. Data are means with sample s.e.m. displayed for 5, 10, 15, 20, 25 and 30 min time points. The numbers of experiments are indicated in parentheses. Letters (a and b) in each time course correspond to the sample currents in the inset. Inset: superimposed currents recorded 5 min after patch break-through. B, I_Ca density in myocytes dialysed with 0.2 mm and 1.8 mm[Mg²⁺]_p in control (0 PP_2A) and low phosphorylation conditions (5 units ml⁻¹ PP_2A) in the presence of 100 nm[Ca²⁺]_p. C, I_Ca density in the rat ventricular myocytes dialysed with 0.2 mm and 1.8 mm[Mg²⁺]_p in control (0 PP_2A) and low phosphorylation conditions (5 units ml⁻¹ PP_2A) in the presence of ∼1 nm[Ca²⁺]_p. Currents were measured 5 min after break-through into the whole-cell patch-clamp configuration. Data are means and s.e.m., with the number of experiments indicated in parentheses. Significant changes of I_Ca, comparing low (0.2 mm) versus high [Mg²⁺]_p (1.8 mm) and basal (0 PP_2A) versus low phosphorylation conditions are indicated as * and ^#, respectively.

In the presence of PP_2A, increasing [Mg²⁺]_p from 0.2 mm to 1.8 mm suppressed peak current by 25 ± 3.4% (n= 10). The magnitude of this effect is statistically smaller than that observed under basal conditions (Fig. 5B). Interestingly, with 1.8 mm[Mg²⁺]_p, I_Ca density at 0 mV was similar with or without PP_2A added to the pipette solution (Fig. 5B). In addition, comparing the time diaries in Figs 1 and 5A shows that channel dephosphorylation had no significant effect on current magnitude in cells voltage-clamped with patch electrodes containing 1.8 mm[Mg²⁺]_p. These results suggested that channel dephosphorylation reduced Mg²⁺ effects on I_Ca.

[Ca²⁺]_i and Mg²⁺ effects on calcium current

The experiments above were performed with [Ca²⁺]_p set to 100 nm, similar to resting levels of [Ca²⁺]_i in rat myocytes. To evaluate the possible influence of [Ca²⁺]_i on [Mg²⁺]_i-dependent modulation of current, I_Ca was measured at different [Mg²⁺]_p in nominally Ca²⁺-free pipette solutions where free [Ca²⁺]_p was approximately 1 nm. Under these conditions, peak I_Ca density at 0 mV in cells voltage-clamped with patch electrodes containing 0.2 mm Mg²⁺ was 14.8 ± 1.1 pA pF⁻¹ (n= 8). Increasing [Mg²⁺]_p from 0.2 mm to 1.8 mm altered I–V relationships and inactivation of I_Ca in a qualitatively similar manner to that with 100 nm[Ca²⁺]_p. However, peak I_Ca amplitude was decreased by 45 ± 2.7% (Fig. 6), a significantly smaller reduction of I_Ca than observed with 100 nm[Ca²⁺]_p.

Figure 6. Effect of [Mg²⁺]_p on I_Ca with different [Ca²⁺]_p.

Currents were measured at a test potential of 0 mV in myocytes dialysed with 0.2 mm and 1.8 mm[Mg²⁺]_p at ∼1 nm, 100 nm and 300 nm[Ca²⁺]_p. Data are means and s.e.m., with the number of experiments indicated in parentheses. Asterisks (*) indicate significant changes of I_Ca between low (0.2 mm) and high [Mg²⁺]_p (1.8 mm).

When cells were voltage-clamped with patch electrodes containing 300 nm Ca²⁺ and 0.2 mm Mg²⁺, peak I_Ca density at 0 mV was 19.4 ± 1.6 pA pF⁻¹ (n= 6). Increasing [Mg²⁺]_p from 0.2 mm to 1.8 mm produced an inhibitory effect on I_Ca quantitatively similar to that observed with 100 nm[Ca²⁺]_p (Fig. 6). These results, summarized in Table 1, suggested that [Ca²⁺]_p did affect [Mg²⁺]_p actions on I_Ca, particularly when [Ca²⁺]_p was decreased to very low levels. The following experiments attempted to define how Ca²⁺ and Mg²⁺ might interact in the cell to modulate I_Ca.

Table 1.

Effects of experimental manoeuvres on [Mg²⁺]_i-dependent modulation of I_Ca

Experimental manoeuvres	Decrease in ICa density (%)†
Patch electrode Ca2+ ([Ca2+]p)
∼1 nm	45 ± 2.7 (8)*
100 nm	64 ± 2.8 (10)
300 nm	71 ± 3.5 (6)
Change cytosolic Ca2+ buffering
10 μm Ryanodine	71 ± 3.8 (5)
5 mm BAPTA + 15 mm EGTA	56 ± 3.5 (6)
Calmodulin blocker
50 μm W7	60 ± 4.9 (5)
Ca2+/calmodulin kinase II blockers
5 μm KN-93	59 ± 5.7 (6)
100 μm AIP	63 (3)‡
Ca2+/calmodulin phosphatase
2B blockers
10 μm CsA	56 ± 5.8 (6)
10 μm CAP	56 ± 2.5 (8)

Values of n given in parentheses. †Percentage decrease of peak I_Ca at 0 mV in cells voltage-clamped with patch electrodes containing 1.8 mmversus 0.2 [Mg²⁺]_p, calculated by Fieller's theorem (Goldstein, 1964). ‡The number of replicates is too small to calculate the mean and 95% Confidence Interval of the ratio, and therefore, the s.e.m. using Fieller's theorem. As a result, the ratio of the mean current density with 1.8 mm and 0.2 mm[Mg²⁺]_i is listed. AIP, autoinhibitory peptide; CAP, calcineurin autoinhibitory peptide.

^*Significantly different from experiments with [Ca²⁺]_p equal to 100 nm.

Effects of intracellular Ca²⁺ fluxes on Mg²⁺ modulation of calcium current

Ca²⁺ influx via l-type channels and sarcoplasmic reticulum (SR) Ca²⁺ release generate local increases in [Ca²⁺]_i (Stern, 1992; Cheng et al. 1993). This local change in [Ca²⁺]_i can significantly modulate both the amplitude and macroscopic inactivation kinetics of I_Ca in ventricular myocytes (Lacampagne et al. 1995; Sham et al. 1995; Qu & Campbell, 1998). To assess how [Ca²⁺]_i participates in the regulation of I_Ca by [Mg²⁺]_i, [Ca²⁺]_i homeostasis was manipulated by two ways: (1) Ca²⁺ release from SR was blocked with 10 μm ryanodine and (2) Ca²⁺ buffering was increased by adding 5 mm BAPTA plus 15 mm EGTA to the pipette solution. In all experiments, [Ca²⁺]_p was set to 100 nm.

In the presence of 10 μm ryanodine, peak I_Ca densities at 0 mV in cells voltage-clamped with [Mg²⁺]_p of 0.2 mm and 1.8 mm were 16.7 ± 1.6 pA pF⁻¹ (n= 7) and 4.5 ± 0.9 pA pF⁻¹ (n= 5), respectively. This change was a 71 ± 3.8% decrease in peak I_Ca amplitude (Table 1), a value not significantly different from that observed without ryanodine. Thus, [Mg²⁺]_p effects on I_Ca were not influenced by SR Ca²⁺ release.

To distinguish between local and global effects of Ca²⁺, slow (EGTA) and fast (BAPTA) Ca²⁺ buffering species were used. Since Ca²⁺ binding kinetics of BAPTA are about 100-fold faster than those of EGTA (Tsien, 1980), Ca²⁺ diffusion distances are quite short (< 100 nm) in the presence of millimolar BAPTA whereas Ca²⁺ diffusion distances can be considerably longer (∼1 μm) in the presence of millimolar EGTA (Allbritton et al. 1992). With 5 mm BAPTA and 15 mm EGTA included in patch pipette solutions, peak I_Ca densities at 0 mV in cells voltage-clamped with 0.2 mm and 1.8 mm[Mg²⁺]_p were 18.9 ± 1.9 pA pF⁻¹ (n= 6) and 7.8 ± 0.9 pA pF⁻¹ (n= 6), respectively. These data show that increasing [Mg²⁺]_p from 0.2 mm to 1.8 mm decreased peak I_Ca amplitude by 56 ± 3.5% (Table 1), a change not significantly different than the [Mg²⁺]_p-induced decrease of I_Ca with 5 mm EGTA (64 ± 2.8%, n= 10). Thus, Ca²⁺ buffers with different kinetics did not affect Mg²⁺ actions on I_Ca.

Effects of Ca²⁺/CaM dependent signal-transduction pathways on Mg²⁺ modulation of Ca²⁺ current

The Ca²⁺-dependence of [Mg²⁺]_i actions on I_Ca, and the inability of Ca²⁺ buffers to alter these actions, point to a possible role for a calmodulin-dependent process in the inhibition of I_Ca. To test this hypothesis, the [Mg²⁺]_i dependence of I_Ca density was examined in the presence of various blockers of calmodulin (CaM) and CaM-dependent enzymes.

In cells voltage-clamped in the presence of the CaM inhibitor, W₇ (50 μm), with pipette solutions containing 0.2 mm[Mg²⁺]_p and 100 nm[Ca²⁺]_p, I_Ca was 6.4 ± 0.8 pA pF⁻¹ (n= 5), a 57 ± 12% decrease compared to I_Ca measured in the absence of W₇. The size of this decrease is consistent with a previous report (Caulfield et al. 1991). Under the same conditions, except that myocytes were exposed to the CaM-dependent protein kinase (CaMKII) inhibitor, KN-93 (5 μm), in the superfusion solution, I_Ca was 9.8 ± 1.2 pA pF⁻¹ (n= 5), a 37 ± 6% decrease compared to I_Ca measured in the absence of this inhibitor. This effect is similar to that reported by Yuan & Bers (1994) with a related CaMKII inhibitor. On the other hand, neither cyclosporin A (CsA, 10 μm in the superfusion solution) nor CAP (10 μm in the pipette solution), inhibitors of CaM-dependent protein phosphatase 2B (PP_2B), produced any significant changes of I_Ca density in myocytes voltage-clamped with pipette solutions containing 0.2 mm[Mg²⁺]_p and 100 nm[Ca²⁺]_p.

Comparing I_Ca density in the presence of these various blockers with 0.2 mm and 1.8 mm[Mg²⁺]_p suggested that a CaM-dependent process was not involved in the [Ca²⁺]_i dependence of [Mg²⁺]_i actions on I_Ca. For example, peak I_Ca at 0 mV in cells voltage-clamped with patch electrodes containing 1.8 mm[Mg²⁺]_p was 3.6 ± 0.7 pA pF⁻¹ (n= 6) and 2.2 ± 0.4 pA pF⁻¹ (n= 5) in the presence of KN-93 and W₇, respectively. These current densities represent a 59 ± 5.7% and 60 ± 4.9% decrease in I_Ca, respectively, when compared to cells voltage-clamped with electrodes containing 0.2 mm[Mg²⁺]_p (see Table 1). This degree of current reduction was not significantly different from that observed in vehicle-control experiments (i.e. a 64% decrease in I_Ca).

Relationship between Ca²⁺ channel phosphorylation and [Ca²⁺]_i on Mg²⁺-dependent reduction of I_Ca

The experiments above suggested that [Mg²⁺]_i-dependent reduction of I_Ca was moderated by reducing channel phosphorylation and [Ca²⁺]_i. The question therefore arises as to whether these two manoeuvres are acting via a common mechanism. To test this possibility, experiments in Fig. 5B were repeated except that pipette solutions were prepared without added Ca²⁺ (i.e. ∼1 nm free [Ca²⁺]_p). Under these conditions and with PP_2A (5 units ml⁻¹) included in pipette solutions, increasing [Mg²⁺]_p from 0.2 mm to 1.8 mm decreased peak I_Ca density from 10.1 ± 1.2 pA pF⁻¹ (n= 6) to 7.0 ± 0.5 pA pF⁻¹ (n= 6), a 23 ± 5.5% (n= 6) decrease (Fig. 5C). In the absence of PP_2A, the degree of current reduction on increasing [Mg²⁺]_p was 45 ± 2.7% (n= 8), significantly different from that observed in the presence of PP_2A. Even so, by comparing Fig. 5B (100 nm[Ca²⁺]_p) and Fig. 5C (∼1 nm[Ca²⁺]_p), it was clear that PP_2A had similar effects on I_Ca amplitude at both low and high [Mg²⁺]_p, irrespective of [Ca²⁺]_p. Thus, these results suggested that increased [Mg²⁺]_i could block the effects of Ca²⁺ channel phosphorylation on I_Ca independently of [Ca²⁺]_i at or below 100 nm.

Discussion

Experiments in this study demonstrated that increasing [Mg²⁺]_p around the reported physiological concentration range, 0.6 and 1.3 mm (Buri & McGuigan, 1990; Hongo et al. 1994), produced a marked inhibitory modulation of l-type Ca²⁺ current, accelerated current inactivation and caused a negative shift in the V_m dependence of current inactivation. Furthermore, manipulating conditions to favour Ca²⁺ channel dephosphorylation, lessened the degree to which [Mg²⁺]_i reduced Ca²⁺ current. This modulation was especially pronounced in the presence of [Ca²⁺]_p (100–300 nm), similar to [Ca²⁺]_i measured in cells at rest. Even so, the dependence of [Mg²⁺]_p effects on channel phosphorylation conditions was largely unchanged over the range of [Ca²⁺]_P tested.

Inhibition of L-type Ca²⁺ current by [Mg²⁺]_i

During the course of whole-cell patch-clamp experiments, I_Ca declined faster and to a much lower level in the myocytes dialysed with 0.6 mm or 1.8 mm Mg²⁺-containing solutions than with solutions containing 0.2 mm Mg²⁺. At the time when current was routinely measured (5 min after break-through), the amplitude of peak I_Ca was 64% smaller in myocytes voltage-clamped with pipette solutions containing 1.8 mm Mg²⁺ than those voltage-clamped with 0.2 mm Mg²⁺, when [Ca²⁺]_p was set to 100 nm with 5 mm EGTA in the patch electrode (Table 1). Previous studies have also shown that elevation of [Mg²⁺] in patch electrode solutions from as low as 1 μm up to 10 mm can dramatically suppress Ca²⁺ current in guinea-pig (Agus et al. 1989; Pelzer et al. 2001; Yamaoka et al. 2002) and frog cardiac myocytes (Yamaoka & Seyama, 1996a,b, 1998; Yamaoka et al. 2002). However, in these previous experiments, changing electrode solution Mg²⁺ concentration around a physiological range of [Mg²⁺]_i produced considerably smaller changes in I_Ca than reported here. These results are, at least in a quantitative sense, different from the present data.

In these previous studies, electrode solutions contained no Mg²⁺ buffers (Agus et al. 1989) or weak Mg²⁺ buffering capacity at physiological [Mg²⁺]_i, i.e. 4 mm ATP (Pelzer et al. 2001) or 3 mm ATP plus 5 mm EDTA (Yamaoka & Seyama, 1996a,b, 1998; Yamaoka et al. 2002). This issue is important because cytosolic Mg²⁺ is largely buffered by proteins and small molecules, such as ATP and phosphocreatine (Robertson et al. 1981; Fabiato, 1983; Konishi & Berlin, 1993) at concentrations that lead one to question the degree to which [Mg²⁺]_i was controlled in previous studies, at least around physiological [Mg²⁺]_i. In contrast, the original report investigating [Mg²⁺]_i regulation of I_Ca in frog myocytes (White & Hartzell, 1988) showed that increasing pipette [Mg²⁺] in the range of 0.3–3.0 mm could significantly decrease I_Ca. In their experiments, pipette Mg²⁺ was buffered by 3 mm ATP and 5 mm phosphocreatine, which leads to a relatively higher Mg²⁺ buffering capacity at physiological concentrations. These results, consistent with the present data where 40 mm Mg²⁺ buffers were present in the patch electrode solution, suggest that changes in [Mg²⁺]_i around a physiological set-point can have large effects in I_Ca.

Pipette solution compositions in this study were chosen to provide high Mg²⁺ buffering capacity at physiological [Mg²⁺]_i, while maintaining MgATP in the millimolar range. Mg²⁺ buffering was provided by 30 mm citrate and 10 mm ATP. Since the dissociation constant of citrate for Mg²⁺ is 0.6 mm (calculated from binding constants in Martell & Smith, 1974), this compound should provide strong buffering through the range of [Mg²⁺]_p used here. A previous report also showed that citrate (10 mm), applied intracellularly, had no effect on I_Ca (Hryshko & Bers, 1992). In any case, total citrate concentration in our pipette solutions was constant so that any citrate effects on I_Ca should have been systematic in this study.

Membrane potential and [Mg²⁺]_i effects on I_Ca

In addition to markedly reducing I_Ca amplitude, increasing [Mg²⁺]_p shifted the I–V relationship by 5–10 mV in the negative direction (Fig. 2A), a finding consistent with Hartzell & White (1989). Likewise, increasing [Mg²⁺]_p from 0.2 to 1.8 mm shifted the V_m for half-maximal current inactivation by –8 mV (Fig. 3). This result, coupled with the acceleration of inactivation of I_Ca by high [Mg²⁺]_p (Fig. 2B), suggests that increasing [Mg²⁺]_p promotes Ca²⁺ channel inactivation. The mechanism responsible for the –8 mV shift in current inactivation was not explored in detail; however, two possibilities are obvious. Cytosolic Mg²⁺ could alter the kinetics of a V_m-dependent gating process and/or change surface charge shielding. Regardless of the particular mechanism, the effect of a –8 mV shift in steady state current inactivation would have only a minor effect on channel availability with a holding potential of –40 mV, as indicated in Fig. 3. Therefore, shifts in V_m-dependent channel gating are unlikely to explain the marked reduction of I_Ca produced by increasing [Mg²⁺]_p.

Channel phosphorylation and [Mg²⁺]_i effects on I_Ca

Channel phosphorylation state appears to exert a strong influence on Mg²⁺ modulation of I_Ca (White & Hartzell, 1988; Agus et al. 1989; Yamaoka & Seyama, 1998; Pelzer et al. 2001). Furthermore, the effects of increasing [Mg²⁺]_i on I_Ca, i.e. decreased amplitude and accelerated inactivation, are consistent with a decrease in Ca²⁺ channel phosphorylation (Allen & Chapman, 1995; Mitarai et al. 2000). Therefore, effects of [Mg²⁺]_i on I_Ca were investigated under conditions strongly favouring or antagonizing l-type channel phosphorylation.

The results of these experiments are quite clear. With 0.2 mm[Mg²⁺]_p, manipulating phosphorylation conditions had a dramatic effect on I_Ca density, ranging from a level of 36 pA pF⁻¹ in the presence of forskolin, IBMX and OA to less than 10 pA pF⁻¹ in the presence of PP_2A. Conversely, at 1.8 mm[Mg²⁺]_p, these same manipulations had little effect on current density. These data, compiled or extrapolated from the experiments, are summarized in Table 2.

Table 2.

Effects of [Mg²⁺]_i on phosphorylation-dependent modulation of I_Ca (pA pF⁻¹)

[Mg2+]p (mM)
Phosphorylation conditions†	0.2	1.8
Low (PP2A)	9.8 ± 1.3 (12)	6.7 ± 0.4 (16)
Basal	17.0 ± 2.2 (12)	5.6 ± 1.0 (10)
High (forskolin, IBMX, OA)	36.3 ± 2.1 (3)	7.6‡

Values of n given in parentheses.

^†[Ca²⁺]_i set at 100 nm and [Ca²⁺]_o to 2 mm in all experiments. Currents were measured at 0 mV.

^‡This value is extrapolated by reducing the current density (36.3 pA pF⁻¹) measured at 0.2 mm[Mg²⁺]_i during superfusion with 2 mm[Ca²⁺]_o by 79%, the reduction in current density measured upon increasing [Mg²⁺]_i from 0.2 to 1.8 mm with 0.5 mm[Ca²⁺]_o.

Viewed in another way, these data suggest that [Mg²⁺]_p has a much greater modulatory role on I_Ca in high phosphorylation conditions (79% reduction) as compared to low phosphorylation conditions (25% reduction). White & Hartzell (1988) also showed that increasing [Mg²⁺]_i ([Mg²⁺]_p ranging from 0.3 to 3 mm) in frog ventricular myocytes had a much greater modulatory effect on I_Ca in conditions promoting Ca²⁺ channel phosphorylation, consistent with our results. Likewise, preincubation of guinea-pig myocytes with a non-specific kinase blocker, K252, to presumably decrease Ca²⁺ channel phosphorylation, abolished any effect of [Mg²⁺]_i on I_Ca when cells were voltage-clamped with pipettes containing solutions in which [Mg²⁺] had been set from 1 μm to 5 mm (Pelzer et al. 2001). These results imply that channel phosphorylation is integral to Mg²⁺ actions on I_Ca.

Why cytosolic Mg²⁺ should produce a greater effect in conditions that promote channel phosphorylation is unclear. One consistent finding is that cytosolic Mg²⁺ inhibits I_Ca with high affinity under basal, presumably low phosphorylation, conditions (IC₅₀= 4 μm; Yamaoka & Seyama, 1996b); however, under conditions promoting Ca²⁺ channel phosphorylation, the apparent affinity for Mg²⁺ inhibition of I_Ca shifts to well over 1 mm (Yamaoka & Seyama, 1998; Pelzer et al. 2001; Yamaoka et al. 2002). This finding might explain why we observe less pronounced effects of [Mg²⁺]_i on I_Ca in the presence of PP_2A, i.e. a [Mg²⁺]_i of 0.2 mm would produce nearly maximal inhibition of I_Ca under dephosphorylating conditions so further increasing [Mg²⁺]_i would have little additional effect on current. Alternatively, micromolar concentrations of GTP are reported to block [Mg²⁺]_i-dependent effects on I_Ca (Yamaoka & Seyama, 1996b; Yamaoka et al. 2002), but channel phosphorylation is reported to overcome these effects of GTP (Yamaoka & Seyama, 1998). Since our pipette solutions contain 0.1 mm GTP, this second possibility seems quite plausible. In any case, our data establish that physiological [Mg²⁺]_i is capable of regulating I_Ca in the presence of GTP to a degree which is dependent on the level of Ca²⁺ channel phosphorylation.

Two types of molecular mechanisms might explain how Mg²⁺ alters gating kinetics of phosphorylated l-type channels. First, the level of channel phosphorylation might be altered because the activity of several regulatory enzymes, such as adenylyl cyclases (Pieroni et al. 1995; Sunahara et al. 1996), phosphodiesterases (Sette & Conti, 1996; Percival et al. 1997) and phosphatases (Cohen et al. 1989; Herzig & Neumann, 2000), are affected by Mg²⁺ at concentrations up to the millimolar range. Second, l-type channel gating has been proposed to be modulated directly by both Mg²⁺ and GTP binding (Yamaoka & Seyama, 1998). Whether one or both of these mechanisms explain Mg²⁺ actions on I_Ca can only be determined by direct measurements of Mg²⁺ effects on Ca²⁺ channel phosphorylation.

[Ca²⁺]_i and [Mg²⁺]_i effects on I_Ca

In the presence of 100 nm[Ca²⁺]_p, increasing [Mg²⁺]_i from 0.2 to 1.8 mm[Mg²⁺]_p suppressed the amplitude of I_Ca by 64%. However, when [Ca²⁺]_p was nominally zero (∼1 nm), increasing [Mg²⁺]_p from 0.2 to 1.8 mm decreased the amplitude of I_Ca by only 45%, a statistically smaller effect. Conversely, when [Ca²⁺]_p was increased to 300 nm, the [Mg²⁺]_i-induced reduction of I_Ca was not different (71%) from that observed with 100 nm[Ca²⁺]_p (Table 1 and Fig. 6). These results suggest that the [Mg²⁺]_p effect is greater in the presence of 100 nm and 300 nm[Ca²⁺]_p than that with ∼1 nm[Ca²⁺]_p and that, to some degree, the effects of [Mg²⁺]_p on I_Ca are achieved in a Ca²⁺-dependent manner, i.e. [Mg²⁺]_i and [Ca²⁺]_i interact to regulate I_Ca.

[Mg²⁺]_i effects on [Ca²⁺]_i regulation of I_Ca

The question then is how [Mg²⁺]_i affects the [Ca²⁺]_i regulation of I_Ca. Our data (Fig. 6) show that increasing [Ca²⁺]_p (1–300 nm) tended to increase I_Ca at low [Mg²⁺]_p (0.2 mm) whereas increasing [Ca²⁺]_p tended to decrease I_Ca at high [Mg²⁺]_p (1.8 mm). These trends in the data suggest that [Mg²⁺]_i might determine the pattern of [Ca²⁺]_i-dependent regulation of I_Ca, e.g. positive or negative regulation of I_Ca.

This latter point was not pursued because, given cell-to-cell variability, a demonstration of statistically significant changes in I_Ca as a function of [Ca²⁺]_i and [Mg²⁺]_i would have required many more experiments. Nonetheless, these results are interesting because the reported effects of increasing [Ca²⁺]_i on I_Ca have varied widely in previous studies. Yamaoka & Seyama (1996a) have reported that increasing [Ca²⁺]_i from 10 nm to 1 μm facilitates I_Ca in frog myocytes voltage-clamped with electrodes containing either 0.1 or 1 mm Mg²⁺. At the higher electrode [Mg²⁺], their results appear opposite of those reported here. No other papers have directly investigated if an interaction between [Mg²⁺]_i and [Ca²⁺]_i might regulate I_Ca. However, many papers have reported that increasing [Ca²⁺]_i can either facilitate (Bates & Gurney, 1993; Gurney et al. 1989; Hirano & Hiraoka, 1994) or inhibit I_Ca and single Ca²⁺ channels (Morad et al. 1988; Hadley & Lederer, 1991; Hirano & Hiraoka, 1994; You et al. 1994). Reviewing these papers does not provide a clear picture about the role of [Mg²⁺]_i in these changes of I_Ca; however, the present results do suggest that changing [Mg²⁺]_i can affect the manner in which [Ca²⁺]_i, around resting levels, might modulate I_Ca.

Possible mechanisms underlying [Ca²⁺]_i modulation of [Mg²⁺]_i effects on I_Ca

To investigate how [Ca²⁺]_i is involved in the modulation of I_Ca by [Mg²⁺]_i, we manipulated [Ca²⁺]_i in two ways: blocking SR Ca²⁺ release with ryanodine, and buffering [Ca²⁺]_i with the fast Ca²⁺ chelator, BAPTA. With 100 nm[Ca²⁺]_p, increasing [Mg²⁺]_p from 0.2 to 1.8 mm decreased I_Ca amplitude to a similar degree in the presence and absence of 10 μm ryanodine, an indication that SR Ca²⁺ release was not involved in [Mg²⁺]_i effects on I_Ca. Furthermore, the [Mg²⁺]_i-induced decrease of I_Ca was similar whether fast (5 mm BAPTA + 15 mm EGTA, 100 nm[Ca²⁺]_p) or slow Ca²⁺ buffer systems (5 mm EGTA, 100 nm[Ca²⁺]_p) were included in the pipette solution. These results indicate that buffering increases of [Ca²⁺]_i, irrespective of the kinetics and capacity of the Ca²⁺ chelator, does not affect Mg²⁺ actions on I_Ca, consistent with our observation that increasing [Ca²⁺]_i with 300 nm[Ca²⁺]_p also does not significantly change [Mg²⁺]_i-dependent modulation of I_Ca. Instead, our data show that [Ca²⁺]_i must be decreased below 100 nm for the interaction between Mg²⁺ and Ca²⁺ to be observed.

Many potential sites exist on or near the l-type Ca²⁺ channel where Ca²⁺ binding could regulate I_Ca (Hering et al. 2000; Herzig & Neumann, 2000). Each of these sites is also likely to be a potential site for Mg²⁺ binding. Even so, our experiments focused on a possible role of calmodulin (CaM) for several reasons. First, CaM can bind both Mg²⁺ and Ca²⁺ (Haiech et al. 1981) and Mg²⁺ binding to CaM interferes with Ca²⁺-dependent regulation of enzyme function (Ohki et al. 1997). Second, the Ca²⁺ affinity of CaM when it is bound to IQ motif peptides is approximately 50 nm (Black et al. 2002), in the same range of Ca²⁺ concentrations used in our pipette solutions. The IQ motif of the l-type channel is located at the C-terminal tail and CaM interaction near this site is thought to participate in channel inactivation and facilitation (Zühlke et al. 1999; DeMarla et al. 2001). Thus, a reasonable expectation is that Mg²⁺ might modulate this Ca²⁺-dependent mechanism of channel gating or vice versa. To test this hypothesis, the effects of CaMKII inhibitors (KN-93 and AIP), calcineurin inhibitors (CsA and CAP) and the CaM inhibitor W₇ were tested on [Mg²⁺]_i-dependent modulation of I_Ca. None of these agents significantly altered the effects of [Mg²⁺]_i on I_Ca. Therefore, our data indicate that a CaM-dependent mechanism does not explain the interaction of [Ca²⁺]_i and [Mg²⁺]_i to modulate I_Ca. Nevertheless, considering that W₇, probably like other calmodulin blockers, may not produce a specific blockade of calmodulin (Klockner & Isenberg, 1987) and Ca²⁺/CaM-dependent inactivation (Imredy & Yue, 1994; Victor et al. 1997), we cannot entirely rule out the involvement of a Ca²⁺/CaM-dependent facilitation/inactivation mechanism in the regulation of I_Ca by [Mg²⁺]_i.

Participation of [Ca²⁺]_i in the phosphorylation-dependent regulation of I_Ca by [Mg²⁺]_i

Since our results show that [Mg²⁺]_i effects on I_Ca amplitude are dependent on channel phosphorylation, we looked at whether this phosphorylation-dependent regulation of Mg²⁺ effects on I_Ca is related in some manner to [Ca²⁺]_i dependence of Mg²⁺ actions. The effect of increasing [Mg²⁺]_p was examined under low and basal phosphorylation conditions with ∼1 nm and 100 nm[Ca²⁺]_p. With both [Ca²⁺]_p, Mg²⁺ effects were comparable, i.e. under basal phosphorylation conditions, increasing [Mg²⁺]_p produced a greater decrease in I_Ca than in the dephosphorylated channel. Most clearly, high [Mg²⁺]_p minimized the effect of channel phosphorylation on I_Ca with both [Ca²⁺]_p. We interpret these data as suggesting that Mg²⁺ can affect two mechanisms, one phosphorylation-dependent and the other Ca²⁺-dependent, that modulate l-type Ca²⁺ channel gating properties.

In summary, the present data show that changes of [Mg²⁺]_p between 0.2 mm and 1.8 mm strongly suppress cardiac I_Ca. These data suggest that cytosolic Mg²⁺ is a potential regulator of I_Ca at physiological concentrations. This modulation of I_Ca by [Mg²⁺]_i is larger in the presence of resting levels of [Ca²⁺]_i, an indication of an interaction between [Mg²⁺]_i and [Ca²⁺]_i. However, Ca²⁺/CaM-dependent signal pathways do not appear to be involved in this modulatory action of [Mg²⁺]_i. Shifts in V_m-dependent gating are also unlikely to be responsible for Mg²⁺ actions. Instead, our results suggest that the channel phosphorylation state plays a predominant role in [Mg²⁺]_i-induced modulation of I_Ca.