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Am J Physiol Heart Circ Physiol 279: H692-H701, 2000;
0363-6135/00 $5.00
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Vol. 279, Issue 2, H692-H701, August 2000

Inhibition by nickel of the L-type Ca channel in guinea pig ventricular myocytes and effect of internal cAMP

Ion A. Hobai, Jules C. Hancox, and Allan J. Levi

Cardiovascular Research Laboratories, Bristol Heart Institute, and Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The characteristics of nickel (Ni) block of L-type Ca current (ICa,L) were studied in whole cell patch-clamped guinea pig cardiac myocytes at 37°C in the absence and presence of 100 µM cAMP in the pipette solution. Ni block of peak ICa,L had a dissociation constant (Kd) of 0.33 ± 0.03 mM in the absence of cAMP, whereas in the presence of cAMP, the Kd was 0.53 ± 0.05 mM (P = 0.006). Ni blocked Ca entry via Ca channels (measured as ICa,L integral over 50 ms) with similar kinetics (Kd of 0.35 ± 0.03 mM in cAMP-free solution and 0.30 ± 0.02 mM in solution with cAMP, P = not significant). Under both conditions, 5 mM Ni produced a maximal block that was complete for the first pulse after application. Ni block of ICa,L was largely use independent. Ni (0.5 mM) induced a positive shift (4 to 6 mV) in the activation curve of ICa,L. The block of ICa,L by 0.5 mM Ni was independent of prepulse membrane potential (over the range of -120 to -40 mV). Ni (0.5 mM) also induced a significant shift in ICa,L inactivation: by 6 mV negative in cAMP-free solution and by 4 mV positive in cells dialyzed with 100 µM cAMP. These data suggest that, in addition to blocking channel conductance by binding to a site in the channel pore, Ni may bind to a second site that influences the voltage-dependent gating of the L-type Ca channel. They also suggest that Ca channel phosphorylation causes a conformational change that alters some effects of Ni. The results may be relevant to excitation-contraction coupling studies, which have employed internal cAMP dialysis, and where Ni has been used to block ICa,L and Ca entry into cardiac cells.

divalent; patch clamp


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE VOLTAGE-GATED L-type Ca channel plays key roles in cardiac muscle (2, 3, 31). It is activated during the upstroke of the cardiac action potential (AP), and inward Ca current through the L-type channel (L-type Ca current; ICa,L) is responsible partly for the characteristic long plateau phase of the cardiac AP (22). Transmembrane Ca entry during flow of ICa,L is thought to be the primary trigger for "Ca-induced Ca release" (CICR) from the sarcoplasmic reticulum (SR) (6, 7) and is an important source of Ca for loading of the SR (3).

Divalent ions are well-known blockers of the L-type Ca channel (21, 34), causing a flicker-type block as they bind to and dissociate from the binding site in the channel pore (21, 34). Divalent ions have been used previously to investigate the role of ICa,L in cardiac muscle (4, 25, 33). Of the different divalent ions, nickel (Ni) may be particularly useful for elucidating the role of Ca entry in cardiac muscle because it blocks both the L-type Ca channel and the Na/Ca exchange (15, 18). However, little is known about the characteristics of Ni block of ICa,L in cardiac muscle (such as the concentration and use dependence and voltage dependence of block), especially at a physiological temperature of 37°C.

beta -Adrenergic stimulation is well known to increase cardiac contractility (3). The resulting increase in cAMP phosphorylates the L-type Ca channel (17), the SR Ca pump (19), and the ryanodine receptor (26), and each of these may play a role in the positive inotropic effect. It has also been suggested that increased cAMP may induce an additional SR release mechanism separate from CICR, which may not require Ca entry (10, 16). In these studies, external Ni was used to block Ca entry. However, a possibility remained that in cells with a raised level of cAMP and phosphorylated Ca channels, the efficacy of Ni for blocking ICa,L might become altered (as has been reported for organic Ca-channel blockers; see Refs. 23 and 27). This provided a second reason for investigating the blocking effect of Ni on the phosphorylated Ca channel.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Myocytes were isolated from the ventricles of guinea pig heart as described previously (16). Cells were kept at room temperature in 1 mM Ca solution until use and usually survived well for up to 8 h.

Electrical recording and rapid solution switches. Cells were placed in a Perspex chamber and superfused at 37°C with Tyrode solution containing (in mM) 140 NaCl, 5 HEPES, 10 glucose, 4 KCl, 2.5 CaCl2, and 1 MgCl2, titrated to a pH of 7.4 with NaOH. After the whole cell configuration had been obtained, the cell superfusate was changed by use of a warmed rapid-switcher device (24). During the experiments, we used a K-free external solution (to inhibit K currents), to which we added different Ni concentrations.

Patch pipettes manufactured from Corning 7052 glass (AM Systems, Everett, WA) were pulled (Micropipette puller, model no. P-87; Sutter Instrument) and fire polished to between 1 and 2 MOmega (Narashige MF 83 microforge). Patch-clamp recordings (12) in the whole cell mode were made with the use of an Axopatch 200A amplifier with a CV202A headstage (Axon Instruments). Compensations were made for cell capacitance and series resistance, and typically we could correct for 70-80% of series resistance.

The basic cesium-based pipette filling solution contained (in mM) 70 cesium aspartate, 40 CsCl, 10 HEPES, 2.5 KH2PO4, 4 MgATP, and 5 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and was titrated to a pH of 7.2 by addition of CsOH. The free Mg concentration was 0.4 mM (a concentration within the normal physiological range; see Ref. 5), and MgATP concentration was 3.6 mM (calculated with the use of Maxchelator for Windows software version 1.2; see Ref. 3). The pipette solution was Na free to attenuate reverse Na/Ca exchange. In some experiments, we added 100 µM cAMP (8-bromo-cAMP; Sigma) to the pipette solution. The "pipette-to-bath" liquid junction potential was -7.5 mV and was corrected before giga-seal formation.

Data analysis and statistics. Voltage-clamp protocols were generated with the use of the program pCLAMP (version 6) via a Digidata 1200A interface (Axon Instruments). Data were recorded on-line and also on a digital data recorder (Instrutech VR-100B, Great Neck, NY), and analysis was performed with the use of pCLAMP6 or AXOSCOPE (version 1.1). Each individual observation was made in myocytes from at least two different guinea pigs to exclude differences that may be due to variations between hearts and cell isolations on each day. Data are expressed as means ± SE, and for statistical analysis we used Student's t-test (variances not assumed equal) and one-way ANOVA. A value of P < 0.05 was taken as significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental protocol. Figure 1A shows the voltage protocol used. From a holding potential of -80 mV, we applied a two-step pulse protocol every 3.25 s; the prepulse depolarization was to -40 mV for 200 ms (to inactivate Na-channel current; INa) and was followed by a 500-ms test pulse to +10 mV. Figure 1A shows records from cells dialyzed with cAMP-free (left) and 100 µM cAMP-containing (right) pipette solution. ICa,L amplitude was measured as the difference between the peak inward current and the current at the end of the 500-ms pulse (17). In this way, the cAMP-activated Cl current (which is known to be time independent; see Ref. 13) did not interfere with ICa,L measurement.


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  		Fig. 1.   A: voltage protocol used and typical membrane currents (Im) in cells dialyzed with cAMP-free (left) and cAMP-containing (right) pipette solution. On average, L-type Ca current (ICa,L) recorded in cells dialyzed with cAMP-free solution peaked in 4.7 ± 0.4 ms and had an amplitude of 11.8 ± 1.1 pA/pF (n = 24 cells). In cells dialyzed with 100 µM cAMP, time to peak was 2.9 ± 0.2 ms, and ICa,L amplitude was 48.6 ± 3.5 pA/pF (n = 30 cells; P < 0.001 for both, unpaired t-test). B: in a cell dialyzed with cAMP-free solution, effect of different Ni concentrations ([Ni]) on ICa,L. C: effect of different [Ni] on ICa,L, in a cell dialyzed with 100 µM cAMP. D: dose-response relationship for Ni on ICa,L amplitude for cAMP-free solution (open circle ) and for 100 µM cAMP (). E: dose-response relation for Ni on ICa,L integral for cAMP-free pipette solution() and solution with 100 µM cAMP (). We identified [Ni] that had a significantly different fractional inhibition of ICa,L in absence and presence of internal cAMP; * P < 0.05.

Dose-response relationship for Ni block of ICa,L. Figure 1B shows that in a cell dialyzed with cAMP-free solution, ICa,L was inhibited in a dose-dependent fashion by Ni. With 5 mM Ni, inhibition was complete and Ni block was rapidly reversible on wash off (within one depolarization; data not shown). In Fig. 1D, we plotted the fractional inhibition of ICa,L as
Fractional inhibition=(<IT>I</IT><SUB>ctr</SUB><IT>−I</IT><SUB>Ni</SUB>)<IT>/I</IT><SUB>ctr</SUB> (1)
where Ictr and INi are ICa,L amplitude in control and during Ni application, respectively. In cells dialyzed with cAMP-free pipette solution (Fig. 1D, open circles), inhibition of ICa,L began around 0.01 mM Ni and was complete between 3 and 5 mM Ni. Data points were curve fitted with a function for cooperative ligand binding (a modified form of the Langmuir-Hill equation; see Ref. 20)
Fractional inhibition=1/{1+(<IT>K</IT><SUB>d</SUB><IT>/</IT>[Ni])<SUP><IT>h</IT></SUP>} (2)
where the dissociation constant (Kd) is the Ni concentration ([Ni]) that gives half-maximal inhibition, and h is the Hill coefficient. Mean results of the fit are shown in Table 1.

                              
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  		Table 1.   Kinetics of Ni block

In addition to peak ICa,L amplitude, we also measured the effect of Ni on the Ca flux via ICa,L by integrating ICa,L for the first 50 ms (the end-pulse current was used as the baseline). Figure 1E illustrates the fractional inhibition of the ICa,L integral by Ni (solid circles); data were fitted as above, and mean results are shown in Table 1.

In cells dialyzed with 100 µM cAMP (Fig. 1C), 0.1 mM Ni had a small inhibitory effect, 0.5 mM inhibited ~50%, and little ICa,L was observed with 5 mM Ni. On average, Ni inhibited ICa,L with a slightly higher Kd (see Table 1 and DISCUSSION), whereas the kinetics of the block of the ICa,L integral were indistinguishable from those in cAMP-free solution (Table 1).

Effect of Ni on time course of ICa,L inactivation. The time course of ICa,L inactivation (with pulses to +10 mV) became changed in the presence of Ni (i.e., Fig. 1C). We fitted ICa,L inactivation with a double exponential function (Fig. 2A) (17)
I<SUB>Ca,L</SUB>(<IT>t</IT>)<IT>=A<SUB>1</SUB> </IT>exp(−<IT>t/&tgr;<SUB>1</SUB></IT>)<IT>+A<SUB>2</SUB> </IT>exp(−<IT>t/&tgr;<SUB>2</SUB></IT>)+C (3)
where A1 and A2 and tau 1 and tau 2 are the maximal amplitudes and time constants of the two exponentials, respectively, and C is the residual current; t is time. In cells dialyzed with cAMP-free solution and in the absence of Ni, tau 1 = 20.80 ± 2.02 and tau 2 = 100.7 ± 7.5 ms (n = 15). The faster component accounted for 0.60 ± 0.06 of total inactivation [calculated as A1/(A1 + A2)]. Figure 2B illustrates the dose-dependent effect of Ni on tau 1 and tau 2. [Ni] between 0.03 and 1 mM had little effect on tau 1 but produced a dose-dependent increase in tau 2. Ni had no significant effect on the fraction of ICa,L inactivation due to each component.


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  		Fig. 2.   A: typical ICa,L in a cell dialyzed with cAMP in control and with 0.3 and 1 mM Ni. Time course of inactivation of ICa,L was fitted with a double exponential (see RESULTS for details). Fit is shown with dashed line. B: effect of Ni on average time constants of the 2 exponentials [tau 1 (open circle ) and tau 2 (); shown as a fraction of control (ctrl), n = 4-6] in cells dialyzed with cAMP-free pipette solution. We could not assess the effect of 3 and 5 mM Ni, because no detectable ICa,L was elicited with these [Ni]. * Significant change in tau  compared with control (P < 0.05). C: as in B, but in cells dialyzed with 100 µM cAMP. D: typical ICa,L elicited in a cell dialyzed with cAMP-free solution for control and 0.1 and 0.5 mM Ni by first (a), fifth (b), and tenth (c) pulse in a train. E: fractional inhibition of ICa,L by 0.1 and 0.5 mM Ni plotted against pulse number, for cells dialyzed with cAMP-free solution. It can be seen that Ni block of ICa,L was, for the most part, not use dependent (1-way ANOVA: P = 1.0 for 0.1 mM Ni, n = 4; P = 0.28 for 0.5 mM Ni, n = 5 cells). There was, nevertheless, a clear trend toward an increase in fractional inhibition with 0.5 mM Ni. Thus we also applied a paired t-test between inhibition achieved with various pulse numbers vs. fractional inhibition achieved during last pulse. F: fractional inhibition of ICa,L, in cells dialyzed with 100 µM cAMP. Effect of 0.1 mM Ni was similar (P = 0.99, 1-way ANOVA, n = 5 cells), and fractional inhibition increased during train for 0.5 mM Ni (P = 0.03, 1-way ANOVA, n = 5 cells). For E and F: * significant difference (P < 0.05). For E and F: open circle , 0.1 mM Ni; , 0.5 mM Ni.

In cells dialyzed with 100 µM cAMP, in the absence of Ni, tau 1 = 10.96 ± 1.30 and tau 2 = 83.44 ± 3.68 ms (P < 0.05 for both, unpaired Student's t-test vs. cAMP-free conditions; n = 15). ICa,L inactivation (0.55 ± 0.02) was due to the faster component (P = not significant vs. cAMP-free solution; n = 15 cells). In cells dialyzed with cAMP, Ni decreased tau 1 and increased tau 2 in a dose-dependent fashion (n = 5-6; Fig. 2C) without changing the proportion of ICa,L inactivation due to each component.

Is Ni block of ICa,L use dependent? Different organic ICa,L antagonists show varying degrees of use dependence (e.g., see Ref. 14); therefore, we investigated the possible use dependence of the Ni block. The protocol used consisted of two parts: 1 min at -80 mV followed by a train of 10 double-step depolarizations (at 0.3 Hz). The protocol was applied first in control and then in the presence of 0.1 or 0.5 mM Ni. We determined the fractional inhibition at each pulse by comparing ICa,L under Ni to ICa,L elicited by the same pulse in control. Figure 2D shows ICa,L elicited (in a cell dialyzed with cAMP-free solution) by the first, fifth, and tenth pulse (respectively) in control solution and with 0.1 and 0.5 mM Ni. If the Ni block of ICa,L was use dependent, then the fractional inhibition obtained during the first pulses should be less than that obtained later in the train. For cAMP-free conditions, 0.1 and 0.5 mM Ni had a substantial effect on ICa,L elicited by the first pulse, and the fractional inhibition remained similar throughout the train (Fig. 2E). If we denote, for each [Ni], the fractional inhibition obtained during the last pulse as maximal, 100% of this was obtained during the first pulse with 0.1 mM Ni and 81% for 0.5 mM Ni. For cells dialyzed with 100 µM cAMP (Fig. 2F), the block by 0.1 mM Ni was constant during the train. Ni (0.5 mM) produced 73% of its maximal inhibition during the first pulse, and degree of inhibition increased slightly during the train. Thus, both in the absence and presence of internal cAMP, the block of ICa,L by 0.1 mM Ni was not use dependent. The block by 0.5 mM Ni seemed to have two components: a large one (73-81%) not use dependent and a smaller use-dependent component (19-27%).

Is Ni block of ICa,L dependent on external Ca concentration? If Ni block of ICa,L involved displacement of Ca from intrapore binding sites, it should be competitive with external Ca concentration([Ca]o). We assessed the fractional inhibition produced by 0.5 mM Ni when we changed [Ca]o from 2.5 to 0.8 and 8 mM. Figure 3A illustrates the typical effect of 0.5 mM Ni (in a cell dialyzed with cAMP) in the presence of 2.5 (top left) and 0.8 mM [Ca]o (top right). Lowering [Ca]o produced a reduction in control ICa,L and a larger inhibition by 0.5 mM Ni. Thus Ni inhibition of ICa,L was facilitated by reducing [Ca]o. Figure 3A, bottom left and right, shows in a different cell that increasing [Ca]o from 2.5 (left) to 8 mM (right) had the opposite effect: inhibition of ICa,L produced by 0.5 mM Ni was decreased. Figure 3B demonstrates that both with and without cAMP, Ni block of ICa,L became larger when [Ca]o was decreased and less when [Ca]o was increased.


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  		Fig. 3.   A: top, in a cell dialyzed with 100 µM cAMP, ICa,L in control and with 0.5 mM Ni in presence of 2.5 (left) or 0.8 mM external Ca (right). Bottom, a similar experiment investigating effect of increasing external Ca from 2.5 (left) to 8 mM (right). B: fractional inhibition of ICa,L by 0.5 mM Ni, plotted against external Ca concentration ([Ca]o) in absence (open circle ) and presence () of internal cAMP. C: Lineweaver-Burk analysis for cells dialyzed with cAMP-free pipette solution. Plot of inverse of effect (1/ICa,L ) against inverse of substrate concentration (1/[Ca]o) in control () and in presence of antagonist (0.5 mM Ni; open circle ). See RESULTS for details. D: as in C, but for cells dialyzed with 100 µM cAMP.

To assess the nature of the relation between external Ca and Ni on ICa,L, we performed a Lineweaver-Burk analysis (Fig. 3, C and D). We plotted the inverse of effect (1/ICa,L) against the inverse of substrate concentration (1/[Ca]o) in control and in the presence of antagonist (0.5 mM Ni), and we fitted regression lines through the points. In the case of a competitive inhibition, where Ca and Ni interact at the same binding site, the two regression lines would intersect on the y-axis. For a noncompetitive effect, where Ni and Ca bind to different sites, which are allosterically linked, a given [Ni] inhibits a certain percentage of ICa,L independently of external Ca, and the two lines would intersect on the x-axis. Both in the absence and in the presence of internal cAMP, Ni block of ICa,L appeared to conform most closely to a competitive model.

Effect of pulse potential on Ni block of ICa,L. We investigated whether Ni block of ICa,L might be voltage dependent, as expected if Ni was an open-channel blocker and as described previously (34). We varied the test potential between -30 and +60 mV, in 10-mV steps (Fig. 4A). This was applied under control conditions and with 0.1 and 0.5 mM Ni. In cells dialyzed with cAMP-free solution, in the absence or presence of Ni, ICa,L reached a maximum between 0 and +20 mV and decreased at more positive potentials (Fig. 4, B and C). We calculated ICa,L conductance (G), as:
G = <IT>I</IT><SUB>Ca,L</SUB>&cjs0823;  (<IT>V</IT><SUB>rev</SUB><IT>−V</IT><SUB>m</SUB>) (4)
where Vrev is the apparent reversal potential (obtained from extrapolating the linear portion of ICa,L decrease at potentials more positive than +20 mV) and Vm is membrane potential. In Fig. 4D, we plotted G normalized for maximum conductance (Gmax) against membrane potential. Data were fitted with a Boltzmann activation curve (17)
<IT>G</IT>&cjs0823;  <IT>G</IT><SUB>max</SUB> = 1&cjs0823;  {1 + exp[(<IT>V</IT><SUB>0.5</SUB> − <IT>V</IT><SUB>m</SUB>)&cjs0823;  <IT>k</IT>]} (5)
where V0.5 and k are the half-maximal activation membrane potential and the slope of the curve, respectively.


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  		Fig. 4.   A: schematic diagram of voltage protocol used. B: in a cell dialyzed with cAMP-free solution, typical ICa,L elicited at different membrane potentials in control and in presence of 0.1 and 0.5 mM Ni. C: voltage dependence of ICa,L in control () and with 0.1 (open circle ) and 0.5 mM Ni (). Data were fitted by eye. D: plot of G/Gmax (where G is conductance and Gmax is maximum conductance) against membrane potential. Mean data from 5 cells were fitted with Eq. 5; fitted parameters are shown in Table 2. To analyze statistically, we fitted ICa,L activation in control and with 0.1 and 0.5 mM Ni in every cell, and we compared groups of fitted parameters (Table 2). Symbols are the same as in C. E: fractional inhibition of ICa,L by 0.1 mM (open circle ) and 0.5 mM Ni () at different membrane potentials. Values for -30 and -20 mV could not be plotted, because control ICa,L was very small. Fractional inhibition was similar for different voltages (P = 0.94 for 0.1 mM Ni and P = 0.09 for 0.5 mM Ni; n = 5). * Voltages where fractional inhibition was significantly different from that obtained at +60 mV (paired t-test, P < 0.05). F: for cells dialyzed with cAMP, voltage dependence of ICa,L in control () and with 0.1 (open circle ) and 0.5 mM Ni (). G: ICa,L activation curve for cells dialyzed with cAMP. Data were fitted with Eq. 5; average results are shown in Table 3. Symbols are the same as in F. H: fractional inhibition of ICa,L by 0.1 (open circle ) and 0.5 mM Ni () was similar for different voltages (P = 0.3 for 0.1 and P = 0.24 for 0.5 mM Ni; n = 4). * Voltages where fractional inhibition was significantly different from that obtained at +60 mV (paired t-test, P < 0.05).

For cells dialyzed with cAMP-free solution, 0.5 (but not 0.1) mM Ni induced a small but significant positive shift in ICa,L activation (Fig. 4D and Table 2). We plotted the fractional inhibition of ICa,L at different potentials (Fig. 4E). The effect of 0.1 mM Ni was independent of pulse potential. The block by 0.5 mM Ni decreased slightly with more positive potentials; however, there was no significant difference in the fractional block between -10 and +60 mV (see DISCUSSION).

                              
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  		Table 2.   ICa,L activation and inactivation parameters for cAMP-free solution

For cells dialyzed with 100 µM cAMP, ICa,L began to activate at -20 mV, reached a peak close to 0 mV (more negative than without cAMP), and decayed at positive potentials (Fig. 4F). A 0.5 mM concentration of Ni (but not 0.1 mM Ni) shifted the peak of the current-voltage curve more positive by ~10 mV. In Fig. 4G, we plotted ICa,L activation variable against membrane potential. Under control conditions, V0.5 was 10 mV more negative, and the slope of the activation was steeper than in the absence of cAMP (Table 3). Ni (0.1 mM) induced a slight negative shift in V0.5, whereas 0.5 mM Ni shifted V0.5 positively. In Fig. 4H, the fractional inhibition of ICa,L with 0.5 mM Ni was larger at -10 mV and fell at more positive potentials. However, for both 0.1 and 0.5 mM Ni, there was no significant difference inhibition at all potentials more positive than 0 mV (see DISCUSSION).

                              
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  		Table 3.   ICa,L activation and inactivation parameters for 100 µM cAMP-containing solution

Effect of prepulse potential on the Ni block of ICa,L. We also investigated whether Ni block was dependent on the availability of the L-type channel (34). To do this, we utilized a protocol in which we varied the prepulse potential. After a two-step protocol (the "reference;" see Fig. 5A), we held the cell membrane at different potentials (between -120 and +5 mV, the "prepulse" potential) for 3 s, after which we applied a depolarization to +10 mV (the "test" pulse). We normalized test ICa,L to the reference ICa,L, to take into account any run down or slow inactivation of ICa,L. TTX (60 µM) was added to inhibit INa. After a run in control solution, we applied a solution containing 0.5 mM Ni and reapplied the protocol.


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  		Fig. 5.   A: schematic diagram of voltage protocol used. B: typical ICa,L elicited from prepulse potentials between -120 and -40 mV in presence of 0.5 mM Ni. Cell dialyzed with 100 µM cAMP. C: test ICa,L (shown as a fraction of reference) in presence of 0.5 mM Ni for cAMP-free solution. There was a small difference (<10% but statistically significant; P = 0.0026, 1-way ANOVA, n = 4), with a smaller ICa,L being recorded at -120 mV. To analyze further, we applied paired Student's t-tests between values from different prepulse potentials compared with -40 mV. * Voltages where difference was significant (P < 0.05). D: in cells dialyzed with cAMP, ICa,L (shown as a fraction of reference) in presence of 0.5 mM Ni against prepulse potential. A 1-way ANOVA test returned P = 0.05, but there was no significant difference between values from any prepulse potential and -40 mV. E: typical ICa,L elicited in control and in presence of 0.5 mM Ni from prepulse potentials between -40 and -10 mV. Cell dialyzed with cAMP-free solution. F: cells dialyzed with cAMP-free solution; effect of Ni on ICa,L voltage dependence of inactivation. Data were fitted with Eq. 6; average results are shown in Table 2. G: cells dialyzed with 100 µM cAMP; effect of Ni on ICa,L voltage dependence of inactivation. Fitted parameters are shown in Table 3. For F and G: , control; open circle , 0.5 mM Ni.

In a first experiment, we varied the prepulse potential between -120 and -40 mV (Fig. 5A). The records are taken from a cell dialyzed with 100 µM cAMP solution. In the absence of Ni, there was no ICa,L inactivation from these potentials (data not shown). Variation of prepulse potential between 120 and -40 mV had little effect on ICa,L recorded in the presence of 0.5 mM Ni. Similar results were observed in cells dialyzed with cAMP-free solution. Mean data (n = 4 cells; Fig. 5, C and D) show test ICa,L (as a fraction of reference ICa,L) plotted against the prepulse membrane potential. The lack of effect on ICa,L amplitude demonstrates that block by Ni was unaffected by prepulse potential.

In a second experiment, we investigated the effect of 0.5 mM Ni on the voltage dependence of ICa,L inactivation. ICa,L elicited from prepulse potentials between -40 and -10 mV, in control and in the presence of 0.5 mM Ni (in the same cell), are shown in Fig. 5E for a cell dialyzed with cAMP-free solution. ICa,L was normalized to the reference, and average data (n = 6) were plotted against prepulse membrane potential and fitted with a Boltzmann equation for steady-state inactivation (Fig. 5F) (17)
I<SUB>Ca,L</SUB><IT> /I</IT><SUB>Ca,L(max)</SUB><IT>=1−1/</IT>{<IT>1+</IT>exp[(<IT>V<SUB>0.5</SUB>−V</IT><SUB>m</SUB>)<IT>/k</IT>]} (6)
where ICa,L(max) is maximum ICa,L. In cAMP-free solution, 0.5 mM Ni shifted the inactivation curve by 5 mV negatively (Fig. 5F and Table 2). Figure 5G illustrates the inactivation curve for cells dialyzed with 100 µM cAMP (n = 7 cells). Under control conditions, cAMP induced a negative shift in V0.5 by 9 mV without a significant change in k (Table 3). In cells dialyzed with cAMP (in contrast to cAMP-free solution), 0.5 mM Ni induced a positive shift in V0.5 by 5 mV (again, with no change in k).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we investigated the characteristics of block of L-type Ca channels by external Ni. We performed experiments both in the absence and presence of dialyzing cAMP to determine whether phosphorylation might alter the properties of Ni block.

Basic characteristics of Ni block. Ni block of ICa,L was rapid. Particularly for high [Ni], such as 3 and 5 mM, block of ICa,L was maximal and complete for the first pulse after application. This suggests that Ni acts at a site on the Ca channel that is close to the external surface or on one that has good access from the external surface. Ni block of ICa,L was also rapidly reversible, with relief from block after wash off largely complete for the subsequent pulse.

Concentration range for Ni block. In cells dialyzed with cAMP-free solution, inhibition of peak ICa,L began to be detectable with 30 µM Ni, and 5 mM Ni produced a maximal inhibition. In the presence of internal cAMP, peak ICa,L was inhibited over a similar [Ni] range; however, the dose-response relation had a slightly higher Kd (0.3 mM for cAMP-free solution vs. 0.5 mM for 100 µM cAMP). We also assessed Ni block of Ca entry via ICa,L by integrating the current during the first 50 ms (which may be more relevant for triggering of CICR; e.g., see Ref. 7). The block by Ni of Ca entry was similar in cells dialyzed with and without internal cAMP, having a Kd close to 0.3 mM. The h close to 1 is at least indicative that there might be one-to-one binding of Ni ions to Ca channels. These data suggest that the fundamental characteristics of Ni block of ICa,L were similar (although not identical) with and without cAMP.

Ni and time course of ICa,L inactivation. We observed that cAMP and Ni each modified the time course of ICa,L inactivation. Internal 100 µM cAMP reduced both tau 1 and tau 2, which agrees with previous studies (29, 30). One explanation might involve the process of Ca-induced inactivation of the Ca channel (9). Raised internal cAMP increases ICa,L magnitude and thus Ca entry. This may be expected to increase the amount of Ca-induced inactivation and would be consistent with both tau 1 and tau 2 becoming shortened in the presence of cAMP. A second possibility is that a phosphorylation-dependent change in conformation might modulate directly the voltage- or Ca-induced inactivation. Both in the absence and presence of internal cAMP, Ni progressively increased tau 2. Because Ni inhibits ICa,L and Ca entry, any Ca-induced inactivation is likely to have become reduced in the presence of Ni, and this may be one mechanism underlying the increase in tau 2 with Ni. We also observed, only in cells dialyzed with 100 µM cAMP, a decrease in tau 1 with Ni. This was in the opposite direction to any increase in tau 1 that might be expected from reduced Ca-induced inactivation of ICa,L. It is possible that the decrease in tau 1 may be consistent with Ni being a fast open-channel blocker (29, 36). However, it is not easy to explain why a decrease in tau 1 was observed only in the presence of internal cAMP, because Ni should also act as an fast open-channel blocker in cells dialyzed with cAMP-free solution.

Competition between Ni and external Ca. Using a [Ni] close to the Kd, we found that block was clearly enhanced in the presence of low Ca and reduced by higher Ca. If Ni and Ca acted at different binding sites, then Ni would have blocked a similar proportion of ICa,L in each different Ca concentration. However, both this behavior and the Lineweaver-Burk plot indicated that the Ni effect on ICa,L was best described by a competitive interaction with external Ca. This suggests that Ni and Ca compete for the same binding site, most probably within the pore of the L-type Ca channel (i.e., see Refs. 34 and 36).

Ni and the activation curve of ICa,L. In the presence of cAMP, the activation curve for ICa,L was shifted negatively in agreement with previous studies (e.g., see Refs. 8 and 28). Both in the presence and absence of cAMP, 0.5 mM Ni induced a positive shift in the ICa,L activation curve. Up to +10 mV, there was a larger fractional block of ICa,L by Ni at negative compared with positive potentials.

This apparent voltage dependence of ICa,L block could be due to the positive shift in the activation curve or else to a voltage-dependent interaction between Ni and a binding site on the L-type Ca channel. In the latter case, with positive potentials repelling Ni electrostatically from a binding site, we might expect to observe a progressive reduction in Ni block over a wide voltage range. However, the fractional ICa,L block became constant at voltages more positive than +10 mV. These data suggest that the positive shift in the ICa,L activation curve by Ni may be the primary reason for the apparent voltage dependence of fractional inhibition. This interpretation agrees with a previous study of the effect of Ni on cloned neuronal Ca channels expressed in Xenopus oocytes (36). However, in a study using cell-attached patches of myotubes from a mouse skeletal muscle cell line (34), it was found that the degree of Ni block increased with larger depolarizations. Possible explanations for these differences are discussed below.

The shift of the activation curve to more positive values by 0.5 mM Ni could be the result of Ni ions binding to a specific site associated with gating of the Ca channel. Alternatively, it could be due to a change in surface charge with Ni (e.g., see Ref. 11). External divalent ions are well known to shift the activation and inactivation curves of Ca, K, and Na channels to more positive potentials (1, 35). These effects have been explained by the adsorption of cations onto negative surface charges at the outer edge of the membrane. This would result in neutralization of these charges and a steeper voltage gradient across the membrane, shifting the gating parameters of ion channels to more positive potentials. Agus et al. (1) found that 1 mM Ni shifted the inactivation curve for the transient outward K current more positive by 5 mV, and this is in reasonable agreement with the 4- to 6-mV positive shifts we found for the ICa,L activation curve with 0.5 mM Ni.

Dependence on holding potential and changes in the inactivation curve for ICa,L. Over the range -120 to -40 mV, which is negative to the range over which ICa,L inactivates, the blocking effect of Ni was independent of the holding potential, both in the absence and presence of cAMP. As mentioned before, Winegar et al. (34) found that block was reduced with hyperpolarization, and this was not evident in the present study (see below).

At potentials more positive than -40 mV, Ni shifted ICa,L inactivation curve, and the direction of movement depended on the presence of cAMP. In cells dialyzed with cAMP, 0.5 mM Ni shifted the ICa,L inactivation curve positively by 4 mV. By itself, this appears consistent with a simple surface charge effect. However, in the absence of cAMP, 0.5 mM Ni reproducibly shifted the inactivation curve by 6 mV in the negative direction. This cannot be explained by an effect of Ni on surface charge and instead suggests that Ni might interact with a specific binding site on the Ca-channel protein concerned with gating. These data show that the effect of Ni on ICa,L is altered by phosphorylation of the channel. At least in the absence of cAMP, a tentative suggestion is that there might be two binding sites for Ni on the Ca channel: one in the ion-channel pore and a second one that modulates voltage-dependent gating.

Comparison with previous studies. The characteristics of Ni block of ICa,L were studied previously by Winegar et al. (34) by use of cell-attached patches of myotubes from a mouse skeletal muscle cell line. The authors recorded (at room temperature) single Ca-channel currents carried by Ba and used the dihydropyridine agonist BAY K-8644 to prolong channel open time. Ni induced a "flicker" type of block. Membrane hyperpolarization increased the rate of unblocking events more than the rate of blocking events, and this led to a reduced affinity for Ni block at negative potentials. Higher concentrations of Ni (>2 mM) also led to a reduction in the amplitude of single-channel current (34). Extrapolation to whole-cell experiments would suggest that the block of ICa,L by Ni might be voltage dependent, with a decreased block at negative potentials. However, we did not observe this in the present study, and instead we found that Ni block of ICa,L was independent of the pulse potential between +10 and +60 mV and also independent of the prepulse potential over the range -120 to -40 mV. The difference is most likely to result from the different experimental conditions. In addition, the cardiac L-type Ca channel has a different structure from the skeletal channel, and the channel may behave differently when high Ba and BAY K-8644 are used together; it is conceivable that either (or both) of these factors may alter properties of Ni block.

Relevance of these results for cardiac excitation-contraction coupling. There are two areas in which Ni block of ICa,L is relevant for cardiac excitation-contraction (E-C) coupling. First, Ni has been used in a number of previous studies to block ICa,L and other Ca entry routes (e.g., see Refs. 25, 32, and 33). However, with hindsight, it seems that Ni was used without basic information on its blocking properties being available. For instance, there were no quantitative data for determining the degree of ICa,L block with a given [Ni] and whether Ni block might be voltage dependent. Our intention was to answer these key questions and so provide a rational basis for the future use of Ni in cardiac cells.

A second important reason for quantitatively assessing Ni block of ICa,L is that this will allow recent (and controversial) results in cardiac E-C coupling to be evaluated objectively. In some recent studies, Ni was applied to cardiac cells dialyzed with cAMP at 37°C (16). The (perhaps surprising) observation was that, in the presence of 5-8 mM Ni, membrane depolarization still elicited SR release, even when Ca entry (and thus CICR) might be expected to be abolished. This raised the possibility of a second, Ca entry-independent, release mechanism in heart cells. One alternative hypothesis was that Ni block of ICa,L might be less efficient in cells dialyzed with cAMP. The present study tested directly this possibility and found no supporting evidence.


    ACKNOWLEDGEMENTS

We thank Lesley Arberry for superb help with the myocyte isolation and Dr. Corne Kros (Univ. of Bristol) for helpful discussions.


    FOOTNOTES

This work was supported by the British Heart Foundation, the Wellcome Trust, the University of Bristol, and the United Bristol Healthcare National Health Service Trust. I. A. Hobai was awarded a postgraduate scholarship by the University of Bristol and an Overseas Research Studentship Award. J. C. Hancox acknowledges the Wellcome Trust for a Research Career Development Award.

Address for reprint requests and other correspondence: I. A. Hobai, Johns Hopkins Univ., Dept. of Medicine, Div. of Cardiology, 720 Rutland Ave., Ross 844, Baltimore, MD 21205 (E-mail: ionhobai{at}welchlink.welch.jhu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 15 July 1999; accepted in final form 8 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(2):H692-H701
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