LVA and HVA Ca2+ Currents in Ventricular Muscle Cells of the Lymnaea Heart

M. S. Yeoman,1,2 B. L. Brezden,1 and P. R. Benjamin1

 1Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG; and  2School of Pharmacy and Biomolecular Sciences, University of Brighton, Moulsecoomb, Brighton BN2 4GJ, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Yeoman, M. S., B. L. Brezden, and P. R. Benjamin. LVA and HVA Ca2+ Currents in Ventricular Muscle Cells of the Lymnaea Heart. J. Neurophysiol. 82: 2428-2440, 1999. The single-electrode voltage-clamp technique was used to characterize voltage-gated Ca2+ currents in dissociated Lymnaea heart ventricular cells. In the presence of 30 mM tetraethylammonium (TEA), two distinct Ca2+ currents could be identified. The first current activated between -70 and -60 mV. It was fully available for activation at potentials more negative than -80 mV. The current was fast to activate and inactivate. The inactivation of the current was voltage dependent. The current was larger when it was carried by Ca2+ compared with Ba2+, although changing the permeant ion had no observable effect on the kinetics of the evoked currents. The current was blocked by Co2+ and La3+ (1 mM) but was particularly sensitive to Ni2+ ions (approx 50% block with 100 µM Ni2+) and insensitive to low doses of the dihydropyridine Ca2+ channel antagonist, nifedipine. All these properties classify this current as a member of the low-voltage-activated (LVA) T-type family of Ca2+ currents. The activation threshold of the current (-70 mV) suggests that it has a role in pacemaking and action potential generation. Muscle contractions were first seen at -50 mV, indicating that this current might supply some of the Ca2+ necessary for excitation-contraction coupling. The second, a high-voltage-activated (HVA) current, activated at potentials between -40 and -30 mV and was fully available for activation at potentials more negative than -60 mV. This current was also fast to activate and with Ca2+ as the permeant ion, inactivated completely during the 200-ms voltage step. Substitution of Ba2+ for Ca2+ increased the amplitude of the current and significantly slowed the rate of inactivation. The inactivation of this current appeared to be current rather than voltage dependent. This current was blocked by Co2+ and La3+ ions (1 mM) but was sensitive to micromolar concentrations of nifedipine (approx 50% block 10 µM nifedipine) that were ineffective at blocking the LVA current. These properties characterize this current as a L-type Ca2+ current. The voltage sensitivity of this current suggests that it is also important in generating the spontaneous action potentials, and in providing some of the Ca2+ necessary for excitation-contraction coupling. These data provide the first detailed description of the voltage-dependent Ca2+ currents present in the heart muscle cells of an invertebrate and indicate that pacemaking in the molluscan heart has some similarities with that of the mammalian heart.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the previous paper Yeoman and Benjamin (1999) described two voltage-gated K+ currents [IK(A) and IK(V)] as part of a study aimed at characterizing ion currents in the myogenic molluscan heart. In this paper, further electrophysiological analysis shows the presence of two voltage-gated calcium currents. We propose that these currents may have a role in pacemaking as well as providing part of the calcium influx necessary for excitation-contraction coupling.

Voltage-gated calcium channels are a common feature of all invertebrate and vertebrate muscles (McDonald et al. 1994). A variety of different Ca2+ currents have been characterized (Kits and Mansvelder 1996). In mollusks, voltage-gated Ca2+ currents have been classified into two distinct groups depending on their voltage sensitivity. Those currents that are activated at relatively hyperpolarized voltage ranges (less than or equal to -40 mV) have been termed low-voltage activated (LVA), whereas those that are activated at potentials more positive to -40 mV have been termed high-voltage activated (HVA). The majority of invertebrate muscles studied to date contain only the HVA type of voltage-activated Ca2+ current. These HVA currents vary slightly in their voltage sensitivity, but all could be blocked with nifedipine. Substitution of Ba2+ for Ca2+ as the permeant ion led to an increase in the amplitude of these currents and a slowing in the inactivation kinetics (Brezina et al. 1994; Laurienti and Blankenship 1996a; Ram and Liu 1991). Consequently, these currents have been classified as L-type. Examples of LVA currents in invertebrate muscle are scarce. The only example to date is from the vas deferens muscle of Lymnaea (Van Kesteren et al. 1995). This current activates at -40 mV, is fast to activate, and has rapid inactivation kinetics. These properties are characteristic of vertebrate T-type Ca2+ currents, although a conclusive pharmacological characterization of this current was not performed. Thus in the invertebrate muscles studied so far, the most prevalent voltage-gated Ca2+ current is the L-type current.

In vertebrates, the situation is more complex. Cardiac, skeletal, and smooth muscle all possess both T- and L-type Ca2+ currents (McDonald et al. 1994). Of the remaining classes of Ca2+ current, neuronal P/Q and R types have no known muscle counterpart, whereas the existence of the N-type channels are extremely rare or absent (Bean 1989b).

In vertebrate cardiac muscle, both T- and L-type calcium currents have been shown to be responsible for action potential generation (Hagiwara et al. 1988). In invertebrate hearts, very little is known about the role of Ca2+ currents in the generation of the myogenic beat. Previous work has shown that the action potentials generated by molluscan heart tissue were strongly dependent on the influx of Ca2+ (Hagiwara and Byerly 1981), but there are no studies where the currents responsible for action potential generation have been characterized. Two types of Ca2+ channel have previously been characterized in dissociated ventricle cells from the Lymnaea heart. These were nonvoltage-gated calcium channels and were initially described by Brezden and Gardner (1990). They were subsequently shown to be activated following the application of the peptide FMRFamide (Brezden et al. 1991). The P(open) time of these channels was extremely low in the absence of the peptide (0.008) (Brezden et al. 1999) and could therefore not be responsible for the generation of the action potentials recorded in dissociated heart muscle cells (Yeoman and Benjamin 1999).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Methods describing the dissociation of heart ventricles and the techniques used to stabilize the cells for intracellular recording are detailed in the previous paper, as are the methods used for data acquisition, analysis and presentation (Yeoman and Benjamin 1999). Ca2+ and Ba2+ currents were isolated pharmacologically (see next section) and leak-subtracted using one of two methods. The first used pClamp's on-line leak subtraction and was based on the resistance of the membrane. The second method involved blocking the isolated Ca2+/Ba2+ currents by substituting the Ca2+/Ba2+ ions in the saline with Co2+ ions. The remaining current was classified as the leakage current. Unless otherwise stated, we have used the latter pharmacological method.

Solutions

To isolate either Ca2+ or Ba2+ currents, the cells were perfused with 30 mM tetraethylammonium (TEA). This was sufficient to block all the K+ currents that normally masked the developing inward currents (Yeoman and Benjamin 1999). These cells do not contain voltage-gated Na+ currents (see RESULTS), so there was no need to include the Na+ channel blocker, tetrodotoxin (TTX), in the bathing solution. The standard solutions used were Ca2+/, Ba2+/, and Co2+/TEA and contained the following concentrations of ions (in mM): 20 Na+, 30 TEA, 1.7 K+, 3.5 Ca2+ or Ba2+ or Co2+, 2 Mg2+, and 10 N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) with the pH adjusted to 7.9 using NaOH. Occasionally, when the Ba2+ currents were difficult to resolve, the concentration of Ba2+ in the extracellular saline was increased to 10 mM. In these instances the concentration of Na+ was reduced to compensate for the increased concentration of Ba2+ ions.

TEA, NiCl2, GdCl3, and LaCl3 were all obtained from Sigma Chemical Co., Poole, U.K., and made up on the day of the experiment by dissolving them in the bath solution. Nifedipine was also obtained from Sigma. A stock solution was made by dissolving the nifedipine in dimethyl sulfoxide (DMSO) and then diluting the solution in the extracellular saline to yield a final concentration of 0.1% DMSO vol/vol. Application of 0.1% DMSO alone was found to have no effect on membrane ion currents. In the experiments that used omega -conotoxin to test for the presence of N-type calcium currents, solutions of the toxin were supplemented with 1 mg/ml of bovine serum albumin to block putative binding sites on the perfusion tubing. All the resulting solutions were adjusted to a pH of 7.9.

Statistical analysis

All values presented in this paper represent means ± SE. Unless stated otherwise in the text, tests for significance have been performed using unpaired t-tests assuming unequal variances (Excel 97).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Initial identification of inward currents

Transient inward currents were observed in ~40% of cells that had been subjected to voltage step protocols in normal saline. These activated at around -70 mV but became masked by the more slowly developing K+ currents at potentials more positive than -40 mV. Ramp protocols showed two regions of negative slope resistance (NSR) typical of the presence of two distinct inward currents. In the example shown in Fig. 1A1, the cell was held at -90 mV and then stepped briefly to -120 mV before being ramped to +40 mV over a 1.25-s period. Although the first phase of inward rectification is clear (I1; -62 mV) the second phase (I2) was almost completely masked by the presence of the voltage-gated K+ currents that began to activate between -50 and -40 mV. To resolve these inward currents more clearly, the same cell was superfused with a bath solution containing 30 mM TEA (Fig. 1A2). This had the effect of blocking the majority of the outward currents and allowed both phases of NSR to be seen more clearly. The first phase of NSR activated around -67 mV and peaked at -60 mV and was provisionally classified as the low-voltage-activated (LVA) current. The second phase activated at around -45 mV and peaked at -25 mV and is referred to as the high-voltage-activated (HVA) current. Although the inclusion of 30 mM TEA in the bathing media allowed us to resolve the inward currents more clearly, there was still a significant outward current present at the more depolarized potentials (more than +10 mV), indicating that not all the outward currents were blocked by TEA.



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Fig. 1. Isolation of voltage-dependent inward currents. A1: voltage ramp protocols (-120 to +40 mV, see bottom) performed on dissociated ventricle cells bathed in normal saline yielded whole cell currents that displayed 2 peaks of inward rectification (I1 and I2). A2: addition of 30 mM tetraethylammonium (TEA) to the bathing solution blocked a large proportion of the K+ currents allowing the 2 inward currents to be more clearly visualized. A3: substitution of the Ca2+ ions in the bathing solution by Ba2+ ions blocked a proportion of the residual K+ currents and further enhanced the amplitude of I2. A4: increasing the concentration of Ba2+ ions to 10 mM blocked the majority of the outward K+ currents and further enhanced the amplitude of I2. B: 2 peaks of inward current were again visible when all the extracellular Na+ ions had been replaced by Ba2+ ions. This indicated that these 2 inward currents are divalent cationic currents. Recordings in A and B are from different cells.

To further characterize these inward currents, Ba2+ ions were substituted for Ca2+ ions in the bathing solution. This had a number of advantages. In the absence of Ca2+ ions, Ba2+ has been shown to enhance the amplitude of a number of types of voltage-gated Ca2+ currents (Bean 1989a; Tsien et al. 1988), as well as being able to block certain K+ currents (Armstrong and Taylor 1980; Hille 1992; Latorre and Miller 1983). Its substitution for Ca2+ in the extracellular solution would help block any residual K+ currents not blocked by TEA and would also prevent the activation of Ca2+-dependent K+ currents also known to be present in these cells. All of these effects would allow the inward Ca2+ currents to be more easily isolated. Figure 1A3 is an example of a current record, from the same cell as in A1 and A2, bathed in a 30-mM TEA/3.5-mM Ba2+ saline with the current evoked using the same ramp protocol. The whole cell current again showed two regions of NSR, indicating the presence of two separate inward currents. As predicted, substitution of Ca2+ with Ba2+ not only reduced the residual outward current at potentials positive to 0 mV but also caused a slight increase in the amplitudes of both inward currents. These two effects were presumably responsible for the shift in the reversal potential of the whole cell current to more positive potentials (from between -10 and 0 mV in 3.5 mM Ca2+ to between +20 and +30 mV in 3.5 mM Ba2+). Increasing the concentration of Ba2+ ions in the saline to 10 mM (Fig. 1A4) further increased the amplitude of the HVA current without significantly altering the amplitudes of either the LVA current or the residual outward current. One unusual, but consistent finding observed when Ba2+ was substituted for Ca2+ in the bathing medium was an atypical shift in the activation threshold of the two phases of inward rectification. Differential effects of the two ions on surface charge screening would normally predict a shift to the left as shown for the voltage step protocols in Fig. 5, and thus we can only assume that this is an artifact seen solely during voltage ramp protocols. The observation that the amplitude of the HVA current alone was dependent on the extracellular concentration of Ba2+ ions suggested that the current was due to the flow of divalent ions and that Lymnaea ventricle cells contained two distinct voltage-gated Ca2+ currents. However, it was possible that the LVA current as well as some component of the HVA current were due to changes in the permeability of the cells to Na+ ions. To test for this, we replaced all the NaCl in the extracellular solution with BaCl2 giving a solution that contained 53.5 mM Ba2+/30 mM TEA. Current records from cells ramped from -120 to +40 mV in this saline showed the same two currents (Fig. 1B), suggesting that Na+ ions were not a significant charge carrier through these channels. The amplitude of both the currents was greater than that seen using 10 mM Ba2+. This was presumably due to the increase in the driving force for Ba2+ entry and indicated that these currents were probably carried by Ca2+ ions in the normal physiological saline. Several other lines of evidence support the hypothesis that both the LVA and HVA currents are Ca2+ currents. First, in five experiments, application of TTX (10-5 M) was unable to block any component of the inward currents (data not shown). Second, in five experiments where all the K+ and Ca2+ currents were blocked pharmacologically by a mixture of TEA and Co2+, we failed to observe any inward current in response to voltage ramp protocols (data not shown). Third, previous work by Brezden and Gardner (1992) failed to show the presence of any voltage-gated Na+ channels using the cell-attached patch-clamp technique in ventricle cells isolated in an identical manner to the present experiments. We therefore believe that Lymnaea heart ventricle cells contain at least two voltage-gated Ca2+ currents.

Variation in the inward current size and the current complement of heart ventricle cells

There were large variations in the size of the inward currents that we recorded. This variation was found between cells taken from the same or different batches of heart muscle cells. In ~60% of batches tested (n = 32 batches), current recordings had to be made using 10 mM Ba2+ as the permeant ion to visualize the currents. In the remaining 40% of the batches (n = 18 batches), the inward currents were large enough to be recorded in normal Ca2+ or Ba2+ salines.

In the majority of fibers recorded (85%; n = 60) two distinct peaks of inward current could be seen (Fig. 2A3). However, a significant proportion of the cells (15%, n = 10) appeared to contain either the LVA current (Fig. 2A1) or the HVA current (Fig. 2A2).



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Fig. 2. Ventricle cells contain a mixture of 2 types of voltage-gated Ba2+ currents. Voltage ramp protocols (-120 to +50 mV) demonstrated that the majority of Lymnaea ventricle cells contained a mixture of both high-voltage-activated (HVA) and low-voltage-activated (LVA) Ba2+ currents (A3). In a minority of cells, only LVA Ba2+ currents (A1), or HVA Ba2+ currents (A2) could be recorded. B1-B3: voltage step protocols were performed on the same cells as A1-A3 to demonstrate the voltage sensitivity and time-dependent kinetics of these currents. C1-C3: I-V plots of the currents recorded in cells B1-B3.

Voltage dependence of LVA and HVA Ba2+ currents

Figure 2, B1-B3, illustrates currents evoked by short (150 ms) voltage steps from -90 mV to potentials between -60 and +30 mV from the same cells as those illustrated in Fig. 3, A1-A3, respectively. Figure 2B1 shows a typical example of the currents evoked by a series of increasingly positive voltage steps from a holding potential of -90 mV in a muscle cell that contained only the LVA Ba2+ current. The LVA current began to activate between -60 and -50 mV (-58 ± 1 mV, mean ± SE, n = 10), peaked at potentials between -40 and -30 mV and then decreased in amplitude at increasingly positive potentials, giving an extrapolated reversal potential of around +40 mV (Fig. 2C1). The HVA Ba2+ current did not begin to activate until -40 or -30 mV (-34 ± 2 mV, n = 8) and peaked at potentials between -10 and 0 mV. It then declined in amplitude, yielding an extrapolated reversal potential of +40 mV (Fig. 2C2). An unpaired t-test showed the activation thresholds of the two currents to be significantly different (P < 0.001). Currents evoked from a cell containing both the LVA and HVA currents are shown in Fig. 2B3. The current-voltage (I-V) plot from this cell showed a characteristic shoulder on its rising phase that corresponded to the peak of the LVA current. Current amplitude continued to increase and peaked at between -10 and 0 mV (the peak of the HVA current). The current then declined to give a predicted reversal potential of around +40 mV (Fig. 2C3).



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Fig. 3. A: variations in the extracellular Ba2+ concentration increases the amplitude of the recorded LVA Ba2+ current but has no effect on its rate of inactivation. B: increasing the concentration of extracellular Ba2+ ions increases the amplitude of the recorded HVA Ba2+ current as well as increasing its inactivation rate. The scaled currents represent the currents obtained in 3.5 mM Ba2+ scaled to the peak of the current obtained in 10 mM Ba2+.

Both inward currents had reversal potentials of around +40 mV. This was far from the theoretical reversal potential for Ba2+ (+175 mV). Similar anomalous reversal potentials for Ba2+ currents have been recorded in other molluscan and vertebrate muscles (Bean 1989b; Brezina et al. 1994). In these other preparations it was proposed that the channels were not perfectly selective for Ba2+ but instead could allow a significant efflux of K+ at depolarized potentials. Permeability to K+ would then tend to shift the reversal potential for the current away from EBa toward Ek.

Activation and deactivation of the LVA and HVA currents

In this and the following section we have quantified the time courses of the activation and inactivation of the two voltage-gated Ba2+ currents.

The LVA current was fast to activate with the time taken for the current to reach its peak decreasing as the potential to which the cell was stepped became more positive. Values ranged from 56.7 ± 8.7 ms with voltage steps to -60 mV (threshold potential) to 17.1 ± 1.5 ms with steps to -40 mV, which activated the peak current (n = 6; Fig. 2B1). Deactivation of the LVA current following the termination of the voltage step was extremely rapid, making a detailed analysis of the tail currents impossible. The time for the HVA current to reach its peak was also rapid and decreased with increasingly more positive voltage steps (Fig. 2B2). The time taken for the current to reach its peak decreased from 44.9 ± 6.7 ms with voltage steps to -40 mV (threshold potential) to 29 ± 2.2 ms with voltage steps to -10 mV, which activated the peak HVA current (n = 6; Fig. 2B2). Like the LVA current, deactivation of the HVA current was again extremely rapid, and the time course of the tail currents could not be resolved with sufficient clarity to perform a detailed analysis. A t-test analysis performed on the time-to-peak for both currents showed them to be significantly different (P < 0.001).

Inactivation of LVA and HVA currents during short voltage steps

As we described earlier, at potentials close to or more positive than those that evoked the largest LVA currents (more positive than -50 mV), the majority of the LVA current (>95%) inactivated completely during a 150-ms voltage step (Fig. 2B1). However, at potentials close to the activation threshold (-70 mV), a component of the LVA current failed to inactivate, leaving a small residual inward current (the possible relevance of this sustained current is detailed further in DISCUSSION). The rate of inactivation of the LVA current was fitted by a single exponential. The rate increased as the membrane was stepped to more depolarized potentials between -60 and -20 mV, indicating a strong voltage dependence (-60 mV; tau  = 59.4 ± 7.6 ms, n = 6; -20 mV, tau  = 15.6 ± 1.6 ms, n = 6; Fig. 2B1). However, at potentials more positive than -20 mV (-20 to +30 mV), the rate of inactivation began to slow (e.g., 0 mV, tau  = 28.9 ± 6.3 ms; n = 6). This indicated that some other mechanism might be important for determining inactivation rates over this potential range. It was possible that this represented some form of current-dependent inactivation. However, there was no correlation between the inactivation rate and the peak amplitude of the current or the amount of current flowing across the membrane (determined by the integral of the current waveform). The most likely explanation was that the inactivation of the LVA current was generally voltage dependent but that the slowing in the inactivation rate at potentials more positive to -10 mV was due to contamination of the LVA current by a small amount of the slower inactivating HVA current. This had previously shown to be maximally active at these potentials.

In contrast to the LVA currents, the HVA Ba2+ currents showed only partial inactivation during the 150-ms voltage step (Fig. 2B2). A double exponential was found to be the best fit for the rate of inactivation. The exponential was fitted to declining current from its peak at the start of the depolarizing voltage step to its final level at the end of the step. Of the two components of the double exponential fit tau 1 was present in the highest proportion (approx 100 times more than tau 2) and was the only component to vary significantly with alterations in the membrane potential of the cell. At potentials just positive to the activation threshold (-20 mV), inactivation was weak (tau 1 = 320 ± 46.3 ms, tau 2 = 15.6 ± 1.6 ms; n = 6). The rate of inactivation increased with more positive voltage steps and became maximal around the peak of the HVA current (0 mV; tau 1 = 116 ± 21.6 ms, tau 2 = 16.5 ± 1.6 ms; n = 6). At potentials more positive than +20 mV, inactivation of the HVA current again became weaker (tau 1 = 280 ± 36.3 ms, tau 2 = 16.1 ± 1.2 ms; n = 6). Using an ANOVA followed by a paired t-test, the values for tau 1 recorded at -20 and +20 mV were found to be significantly different to those at 0 mV. The inactivation of the Ba2+ HVA current therefore does not appear to be only voltage dependent but may also be current dependent.

This was examined in experiments where inactivation of the HVA Ba2+ current was recorded in salines where the extracellular concentration of Ba2+ ions had been increased from 3.5 to 10 mM to increase the current flow through the HVA channels. Figure 3B shows an example of one such experiment where HVA currents were evoked by 150-ms voltage steps to +10 mV from a holding potential of -90 mV. Increases in the concentration of divalent ions bathing the cells clearly increased the rate of inactivation of the HVA current confirming a current-dependent mechanism of inactivation. Again tau 1 was the only time constant to be significantly altered. tau 1 for currents recorded in 3.5 mM Ba2+ was 235 ± 19.5 ms (n = 6). This value decreased to 130.3 ± 4.7 ms for currents recorded in 10 mM Ba2+ (paired t-test, P < 0.001). In contrast the rate of inactivation of the LVA Ba2+ currents recorded in the same experiments was not significantly affected by changes in the extracellular concentrations of divalent ions (Fig. 3A), confirming that inactivation of the LVA current is not current dependent.

The values of the time constants detailed above provide further information confirming the distinctness of the two currents. However, because of our inability to completely isolate either of the two currents, these values may not represent the true kinetics of the pure currents.

Ca2+ and Ba2+ permeability of the LVA and HVA currents

Based on the voltage sensitivity and time-dependent kinetics of the LVA and HVA currents, these two currents appeared to be members of the T-type and L-type family of Ca2+ currents, respectively. Further evidence in support of this classification would be obtained by examining the relative permeabilites of the two currents to Ca2+ or Ba2+ ions. T-type channels have previously been shown to have no preference for Ca2+ ions over Ba2+ ions (Alvarez and Vassort 1992; Bean 1985; Chesnoy-Marchais 1985; Coyne et al. 1987). L-type channels on the other hand have been shown, in a variety of invertebrate and vertebrate muscles, to conduct Ba2+ ions much better than Ca2+ (Bean 1989b; Tsien et al. 1988). We have therefore examined the relative amplitudes of the LVA and HVA currents in Lymnaea ventricle cells in the presence of equimolar (3.5 mM) physiological concentrations of either Ca2+ or Ba2+. Figure 4 shows examples from three different cells of both LVA (A1-A3) and HVA currents (B1-B3) recorded in the two different salines.



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Fig. 4. Permeability of LVA and HVA currents to Ca2+ and Ba2+ ions. A1-A3: LVA currents evoked from 3 separate cells by voltage steps to -50 mV from holding potentials of -90 mV in salines containing either 3.5 mM Ca2+ or 3.5 mM Ba2+ ions. B1-B3: HVA currents evoked from the same 3 cells as A1-A3, by voltage steps to -10 mV from a holding potential of -90 mV. Currents were again recorded with either 3.5 mM Ca2+ or 3.5 mM Ba2+ ions as the permeant ion.

LVA currents were evoked by a 200-ms voltage step from -90 to -50 mV. In 3.5 mM Ca2+ they were fast to activate and in the majority of cases (12 of 13 cells tested) inactivated completely during the 200-ms voltage step (Fig. 4, A1 and A3). In the remaining cell tested, a sustained inactivating component of the LVA Ca2+ current was present (Fig. 4A2). Due to the infrequent appearance of this current, we have not been able to determine whether or not this current is different from the more typical LVA current recorded in the majority (92%) of cells. Substitution of Ba2+ for Ca2+ caused a marked reduction in the amplitude of the LVA current in the majority of fibers tested 50.6 ± 10.6% (mean ± SE; in 12 of the 13 fibers tested, Fig. 4, A1 and A3). This demonstrated, that on average, LVA channels were more permeable to Ca2+ than Ba2+ ions. In one cell only, LVA currents were recorded that showed equal permeability to Ca2+ and Ba2+ (Fig. 4A2). It is interesting to note that this was seen in the cell that displayed the noninactivating current.

HVA currents were evoked by voltage steps from -90 to -10 mV. The HVA Ba2+ currents were typically larger than the corresponding LVA currents recorded from the same cells. They were all fast to activate but showed varying rates of inactivation (compare Fig. 4, B1 and B3 with B2). Compared to the Ca2+ currents recorded from the same cells, HVA Ba2+ currents were noticeably larger (48.6 ± 6.7% mean ± SE; 10 of 13 cells tested). This indicated that these channels conduct Ba2+ better than Ca2+. In the other three cells tested, one showed no increase, whereas the others showed decreases of 156 and 242%, respectively. HVA Ca2+ currents like the Ba2+ currents were fast to activate but showed complete inactivation during the 200-ms voltage step. The inactivation rates of the HVA Ca2+ currents were fitted best with a double exponential and were therefore similar to the HVA Ba2+ currents described earlier. However, unlike the HVA Ba2+ currents the proportions of the two components comprising the inactivation phase of the HVA Ca2+ currents are approximately equal. Changing the permeant ion caused significant changes in the first time constant (tau 1) from 81.6 ± 11.3 ms (Ca2+) to 364.2 ± 46.4 ms (Ba2+; n = 8, P < 0.001), whereas there were no changes in tau 2 = 16.6 ± 1.5 ms (Ca2+) to 15.5 ± 1.8 ms (Ba2+). The faster inactivation rate seen in cells bathed in the Ca2+-containing saline could be due to the activation of a Ca2+-dependent K+ current, that has been shown to be present in other molluscan muscles (Brezina and Weiss 1995; Laurienti and Blankenship 1996b), but this has not been examined in the present experiments.

The changes in conductance that we have recorded for both LVA and HVA currents in the Ca2+ and Ba2+ salines are consistent with these two currents being members of the T- and L-type calcium channel families, respectively.

Comparison of the voltage dependence of LVA and HVA currents using normal concentrations (3.5 mM) of Ca2+ or Ba2+ as the permeant ions

It was important to generate I-V curves for the HVA and LVA currents at concentrations of Ca2+ resembling those present in normal blood (3.5 mM) and with similar concentrations of Ba2+ ions, for comparison. The function of the currents could then be discussed in relation to the known operating range of voltages seen in previous recordings of beating activity in the isolated cells (Yeoman and Benjamin 1999) carried out in normal saline. Figure 5 shows a series of current recordings from a single cell in normal saline with either Ca2+ (Fig. 5A) or Ba2+ (Fig. 5B) as the permeant ion. In Fig. 5A1 Ca2+ currents were evoked by holding the cell at -90 mV and stepping it to a range of potentials between -80 and +40 mV. An I-V plot of the total inward currents are shown in Fig. 5A4 (). The whole cell inward current was seen to activate between -75 and -65 mV and showed an initial peak around -50 mV (LVA current) and a second larger peak of inward current between -10 and 0 mV (HVA current). By holding the cell at more depolarized potentials (-50 mV; Fig. 5A2), it was possible to inactivate all the LVA current so that the same series of voltage steps (-80 to +40 mV) now evoked an almost pure HVA current. This current activated around -40 mV (-42 ± 2 mV; n = 8) and peaked between -10 and 0 mV before declining in amplitude and yielding an extrapolated reversal potential of around +50 mV (, Fig. 5A4). By subtracting the currents recorded in Fig. 5A2 from those recorded in Fig. 5A1 we were able to isolate the LVA current (Fig. 5A3). This current activated between -75 and -65 mV (-72.0 ± 12 mV; n = 10) and peaked at between -50 and -40 mV before declining in amplitude and showing an apparent reversal potential around +50 mV. The small inflection seen at -20 mV probably represented a small proportion of the HVA current that is also blocked by holding the cell at -50 mV. Figure 5B shows a series of current records from the same cell as that recorded in Fig. 5A, but this time superfused with a 3.5 mM Ba2+ saline. Currents were again evoked from holding potentials of -90 mV (Fig. 5B1) or -50 mV (Fig. 5B2) with LVA currents again isolated by subtracting currents recorded from a holding potential of -50 mV from those evoked from a holding potential of -90 mV (Fig. 5B3). LVA currents recorded under these conditions were again fast to inactivate (Fig. 5B3). The HVA currents on the other hand showed the similar slow inactivation rates that we had observed previously (see Fig. 4). The I-V plots of the isolated currents are shown in Fig. 5B4. LVA currents recorded in 3.5 mM Ba2+ were extremely small and difficult to resolve due to the relatively low permeability of the channels to Ba2+ (see Fig. 4). At potentials more positive than -30 mV, their small size meant that there was significant contamination of this current from the small proportion of HVA current that was also blocked by holding the cell at -50 mV (black-lozenge , Fig. 5B4). Because of their small size we were unable to obtain reliable data for the voltage dependence of the LVA Ba2+ current recorded under these conditions. HVA currents on the other hand activated between -50 and -40 mV, peaked at potentials between -20 and -10 mV, and reversed around +30 mV. This negative shift in the I-V curve for the HVA Ba2+ current compared with the HVA Ca2+ current is probably due to the fact that Ca2+ is a better screen of the surface membrane charge than equimolar concentrations of Ba2+ (Hille 1992).



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Fig. 5. Comparison of the voltage sensitivities of LVA and HVA currents using 3.5 mM Ca2+ or 3.5 mM Ba2+ as the permeant ion. A1: whole cell LVA and HVA Ca2+ currents evoked by voltage steps from a holding potential of -90 mV. A2: whole cell HVA Ca2+ currents evoked by voltage steps from a depolarized holding potential (-50 mV) designed to inactivate the majority of the LVA current. A3: currents recorded in A1 minus A2 yield a net LVA current. A4: I-V plots of whole cell (), LVA (black-lozenge ), and HVA Ca2+ currents (). B1: HVA and LVA Ba2+ currents evoked from holding potentials of -90 mV. B2: HVA Ba2+ currents evoked from a holding potential of -50 mV that inactivates the majority of the LVA current. B3: subtraction of currents recorded in B1 minus B2 to yield pure LVA Ba2+ current. B4: I-V plot of the whole cell (), LVA (black-lozenge ), and HVA Ba2+ currents (). Recordings in A and B are all from the same cell.

Steady-state inactivation of HVA and LVA currents

The resting membrane potential of Lymnaea heart ventricle cells was previously shown to be about -55 mV (Brezden and Gardner 1986; Buckett et al. 1990; Yeoman and Benjamin 1999). An analysis of the I-V relationship of the LVA current detailed in the previous section indicated that from holding potentials of -90 mV there was a significant activation of this current between -70 and -60 mV, suggesting that this current could contribute significantly to the depolarization that drives spontaneous spiking activity in the cells (Yeoman and Benjamin 1999). However, steady-state inactivation at normal membrane potentials might make the contribution of the LVA current ineffective. This was examined by holding cells at a variety of membrane potentials (between -110 and -20 mV) and stepping to -50 mV to activate the LVA current. The experiments used 3.5 mM Ca2+ as the permeant ion. The amplitude of LVA currents was unaffected by holding potentials more negative than -70 mV. As the holding potential was made more positive than -70 mV, the amplitude of the evoked current declined and was abolished completely by holding potentials more positive than -40 mV (Fig. 6A1). The data from three cells showing the effect of holding potential on the amplitude of the evoked current is plotted in Fig. 6A2. The three sets of data could be fitted with a Boltzmann curve that showed that the LVA current was half inactivated at -58 mV and had a slope factor of 4.1 ± 0.3 mV. Due to the steep slope of the inactivation curve, only about one-third of the LVA current would be available at -55 mV, the resting membrane potential of the cell, but could still contribute to the pacemaker activity of the isolated heart muscle cells (see DISCUSSION).



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Fig. 6. Holding potential dependence of LVA and HVA Ca2+ currents. A1: LVA currents evoked from a variety of different holding potentials (-110 to -30 mV, see marked potentials) by test potentials to -50 mV. A2: plot of holding potential dependence of LVA Ca2+ currents from 3 different cells (different symbols). Solid line is a Boltzmann fit of the 3 sets of data. Dotted line represents the potential at which 50% of the current is inactivated. B1: HVA currents evoked from a variety of different holding potentials (-110 to -20 mV, see marked potentials) by test potentials to -10 mV. B2: plot of holding potential dependence of HVA Ba2+ currents from 3 different cells (different symbols). Solid line is a Boltzmann fit of the 3 sets of data. Dotted line represents the potential at which 50% of the current is inactivated.

HVA currents were recorded using 3.5 mM Ba2+ as the permeant ion and were activated by short 150-ms voltage steps from a variety of holding potentials (-110 to -20 mV) to 0 mV. We chose not to use Ca2+ as the permeant ion because of the risk of contaminating the evoked Ba2+ current with unblocked K+ currents that would be significantly activated at this potential. Unlike the LVA current, the HVA current was relatively unaffected by holding potentials as positive as -60 mV. At potentials more positive than this, the current again declined in amplitude and was abolished completely by holding potentials of around -20 mV. Sample current traces of HVA currents evoked from a variety of holding potentials are shown in Fig. 6B1. The combined data from three such experiments is plotted in Fig. 6B2 and again has been fitted with a Boltzmann curve, indicating that the HVA current is half inactivated at a holding potential of -48 mV and had a slope factor of 4.1 ± 0.3 mV. Thus, although a significant proportion of this current is available for activation at the RMP of the cell, its relatively positive activation threshold (-40 mV) means that this current cannot contribute to the initiation of pacemaking in these cells, but could contribute to the action potentials once they had been triggered.

Pharmacological profile of LVA and HVA currents

As in other systems La3+, Gd3+, and Co2+ produced 100% block of both LVA and HVA currents at 3.5 mM, whereas a similar block could be obtained using 1 mM Cd2+ (data not shown). These data are in agreement with previous reports and provide further evidence that both the LVA and HVA currents are carried predominantly by Ca2+ ions. Here we were mainly interested in characterizing compounds that were capable of selectively blocking one or other of the two Ca2+ currents. Previous work has shown that the dihydropyridine compounds such as nifedipine are capable of selectively blocking L-type calcium currents (Bean 1989a; Brezina et al. 1994; Tsien et al. 1988); so if the HVA current in Lymnaea ventricle cells was sensitive to nifedipine, it would provide further evidence that this current was a member of the L-type calcium channel family. Figure 7 shows an example of such an experiment in which the sensitivities of both LVA and HVA currents to 10 µM nifedipine were examined. In this experiment, currents were recorded in fibers bathed in the elevated Ba2+ solution (10 mM). LVA and HVA currents were evoked by holding the fiber at -90 mV and stepping it to -40 and 0 mV, respectively (these were the peak potentials for these currents in the elevated Ba2+ saline). Figure 7A1 shows a record of an HVA current evoked in normal saline (control) or in the presence of 10 µM nifedipine. The current in nifedipine is markedly reduced, indicating that a significant proportion of the current is sensitive to this dihydropyridine compound. Interestingly, the block was more potent toward the end of the voltage step (57.3 ± 5.6%; n = 8; P < 0.01) compared with the instantaneous block (32.4 ± 4.74%; n = 8; P < 0.01). This is in agreement with previous studies that showed that nifedipine antagonized the channel by means of an open channel block (Bean et al. 1986; Carbone and Swandulla 1989). We investigated this possibility by prepulsing the fibers to +50 mV to maximally activate the HVA channels and then tested the block by nifedipine. Figure 7C shows a comparison of the current blocked by 10 µM nifedipine using the standard protocol (Fig. 7A) versus a protocol where the cell was prestepped to +50 mV. Prepulsing the cell to +50 mV can clearly be seen to facilitate the block of the HVA current by nifedipine. In three preparations the instantaneous current was blocked by 68.8 ± 7.8% at the end of the voltage pulse during the standard protocol compared with the 86.2 ± 9.3% (mean ± SE) seen with the prepulse. T-type calcium channels, on the other hand have previously been shown to be insensitive to concentrations of dihydropyridines that produce a significant block of L-type channels (Bean 1985; Hagiwara et al. 1988). Consistent with this was our observation that nifedipine had no significant effects on the amplitude of the LVA current (P > 0.05; Fig. 7A2). The I-V relationship of peak current evoked from the cell recorded in Fig. 7A in the presence and absence of nifedipine is illustrated in Fig. 7B. The figure clearly shows that nifedipine blocks a current that activates between -30 and 0 mV, the voltage range where the HVA current is maximally active. The lack of block at potentials more positive than 0 mV indicates that the block may show some voltage dependence.



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Fig. 7. Nifedipine sensitivity of LVA and HVA Ba2+ currents. A1: HVA Ba2+ currents evoked by voltage steps from -90 to 0 mV were significantly reduced following the application of 10 µM nifedipine. A2: LVA Ba2+ currents evoked by voltage steps from -90 to -40 mV were unaffected by 10 µM nifedipine. B: I-V plot of whole cell Ba2+ currents in the presence () and absence () of 10 µM nifedipine. C: blockade of the HVA Ba2+ current is enhanced in cells that were prepulsed to +50 mV compared with control currents evoked in the absence of a prepulse.

The HVA current in Lymnaea heart ventricle cells required approximately a 10-fold higher concentration of nifedipine to produce the same %block when compared with the L-type current in another invertebrate muscle (ARC muscle) (Brezina et al. 1994). This raised the possibility that the HVA current in Lymnaea ventricle cells may be made up of more than one current type. One possibility was that the HVA current we were recording consisted of a mixture of N- and L-type currents. N-type channels like L-type are another example of HVA currents. However, they can be specifically blocked by omega -conotoxin GVIA (Hille 1992). In four experiments of the sort described above, application of 10 µM omega -conotoxin GVIA failed to cause any significant block of the HVA current. This indicated that the HVA current in Lymnaea ventricle cells appeared to consist of a homogenous population of L-type channels that was relatively insensitive to nifedipine.

In contrast, T-type channels in muscles have been shown to be sensitive to micromolar concentrations of Ni2+ (Bonvallett 1987; Hagiwara et al. 1988). We therefore examined the ability of Ni2+ to selectively block the LVA channel. In Fig. 8A1 application of 100 µM Ni2+ produced a significant block of the LVA channel (38.9 ± 2.07%; n = 8; P < 0.01). At these concentrations Ni2+ did not affect the amplitude of the HVA current (Fig. 8A2). An example of the current-voltage relationship of the same cell in the presence and absence of 100 µM Ni2+ is shown in Fig. 8B. In the presence of the Ni2+ ions, there is a marked reduction in the current flowing between -60 and -40 mV, where the LVA current is maximally active. Increasing the concentration of Ni2+ to 1 mM completely blocked the LVA current but also blocked over 80% of the HVA current (data not shown). Other potential blockers of LVA currents such as amiloride (Tytgat et al. 1990) showed no selectivity for the LVA over the HVA current (data not shown).



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Fig. 8. Ni2+ sensitivity of LVA and HVA Ba2+ currents. A1: LVA Ba2+ currents evoked by voltage steps from -90 to -40 mV were markedly reduced following the superfusion of 100 µM Ni2+. A2: HVA Ba2+ currents evoked by voltage steps from -90 to 0 mV were not significantly affected following superfusion with 100 µM Ni2+. B: I-V plot of whole cell Ba2+ currents in the presence () and absence () of 100 µM Ni2+. Arrow indicates the region of the I-V curve where Ni2+ causes a marked reduction of the whole cell current. Recordings in A1 and A2 and data plotted in B are from the same cell.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Lymnaea heart ventricle cells contain two types of voltage-gated Ca2+ currents

Ramp protocols, performed on voltage-clamped Lymnaea heart ventricle cells bathed in normal saline, indicated the presence of two distinct inward currents. These currents have been termed LVA and HVA currents based on a classification by Skeer et al. (1996). These currents could be isolated by perfusing the ventricle cells with TEA to block the voltage-gated K+ currents (Yeoman and Benjamin 1999). Further more detailed electrophysiological and pharmacological analysis of the two currents, mainly using step voltage clamp protocols, led us to identify them as members of the T- and L-type families of Ca2+ currents, which have been extensively studied in vertebrates and are present in a wide variety of different muscle types (McDonald et al. 1994).

The Lymnaea LVA Ca2+ current activated at potentials between -75 and -65 mV, peaked between -50 and -40 mV, and reversed between +30 and +40 mV. This low threshold for activation is characteristic of LVA and T-type Ca2+ currents in both invertebrates (Kits and Mansvelder 1996) and vertebrates (Alvarez and Vassort 1992; Hagiwara et al. 1988; McDonald et al. 1994). The current was fast to activate and at potentials above -50 mV showed complete inactivation during short (200 ms) voltage steps. The time constant for inactivation appeared to be voltage dependent, which was again consistent with data obtained from T-type currents in vertebrate atrial (Bean 1989a), smooth (Akaike et al. 1989), and skeletal muscles (Cognard et al. 1986). The LVA current also showed progressive steady-state inactivation as the holding potential from which it was evoked was made more positive. The LVA current was half inactivated at -58 mV and completely inactivated at holding potentials more positive than -40 mV. These values were consistent with those obtained for T-type currents recorded from a variety of muscles types and neurons (McDonald et al. 1994). However, direct comparisons were extremely difficult as the majority of T-type currents were extremely small and were therefore recorded using elevated, rather than normal concentrations of the divalent ions.

An examination of the relative permeabilities of the LVA channel to normal levels of Ca2+ or Ba2+ (3.5 mM) showed that, in the majority of cells tested, LVA channels were more permeable to Ca2+ than Ba2+ ions. The majority of T-type channels in muscle cells appear to show little or no preference for either Ca2+ or Ba2+ as a permeant ion (Alvarez and Vassort 1992; Bean 1985; Chesnoy-Marchais 1985; Coyne et al. 1987), and therefore in this respect the LVA channel was different from those previously described in the vertebrate literature. Lymnaea ventricle LVA currents were also sensitive to block by extracellular Ni2+ ions (100 µM; approx 50% block). Sensitivity to Ni2+ ions is another characteristic of T-type calcium currents (Bonvallett 1987; Hagiwara et al. 1988). On average vertebrate T-type currents are completely blocked by 40 µM Ni2+ (Hagiwara et al. 1988) and are therefore much more sensitive than the Lymnaea current. However, the Ni2+ sensitivity of the Lymnaea LVA current is comparable to other invertebrate LVA currents, which were also relatively insensitive to Ni2+ (Kiss and Osipenko 1991). These data therefore provide convincing evidence that the LVA current in Lymnaea ventricle cells is a member of the T-type family of Ca2+ currents.

The HVA current that we have characterized has a number of properties that resemble those of previously described L-type Ca2+ currents. With normal levels of extracellular Ca2+ (3.5 mM) the activation threshold was ~30 mV more positive than that for the LVA current (-40 to -30 mV) and peaked at potentials between -10 and 0 mV. This was comparable to values in invertebrate neurons (Byerly and Hagiwara 1982; Eckert and Ewald 1983) and vertebrate muscle and neurons (reviewed by Bean 1989b). In extracellular solutions containing physiological levels of Ca2+, the HVA current was fast to activate and appeared to inactivate completely during standard 200-ms voltage steps. In equimolar Ba2+ solutions, HVA currents were again fast to activate but exhibited much slower rates of inactivation during standard 200-ms voltage steps. Previous work on both HVA and L-type currents in other systems has demonstrated similar differential effects of Ca2+ and Ba2+ ions on inactivation (Eckert and Chad 1984). This change in the inactivation rate could be due to a number of factors. First, under physiological conditions the inactivation of the HVA and L-type currents has been shown to be strongly dependent on the intracellular concentration of Ca2+ ions and only weakly voltage dependent (i.e., that portion of the inactivation remaining in currents recorded in Ba2+-containing solutions). In a given extracellular solution the rate of inactivation of the HVA currents recorded at a variety of different membrane potentials showed no correlation to the voltage at which the cell was being held but was more closely related to the size of the evoked current. Thus at physiological concentrations of extracellular ions, this would be related to the amount of Ca2+ entering the cell. However, current-dependent inactivation is also seen with Ba2+ as the charge carrier, although the responses of the cell are greatly attenuated, indicating that the channels are more sensitive to Ca2+. This conclusion was further substantiated by the observation that manipulations designed to alter the size of the current that flowed through HVA channels (increasing the extracellular concentration of divalent ions) had the appropriate effects on the inactivation rates. Equally, the increased inactivation observed with Ca2+ as the permeant ion could be an artifact due to residual unblocked Ca2+-dependent K+ currents that would not be activated in Ba2+-containing solutions (Hermann and Gorman 1979). Similar findings have been described in other invertebrate preparations (Brezina et al. 1994). The Lymnaea HVA current also showed marked steady-state inactivation when the voltage from which the current was evoked was made more positive. Typically the HVA current was half inactivated at -48 mV and completely inactivated at holding potentials more positive than -30 mV. Again these data are consistent with those obtained in other invertebrate and vertebrate preparations (Brezina et al. 1994) (half inactivation -45 mV). Further confirmation that the HVA current we had characterized was a member of the L-type Ca2+ current family was its block by the dihydropyridine (DHP), nifedipine. The DHP binding site has previously been shown to be a part of the L-type Ca2+ channel, and therefore the block of our HVA current by nifedipine confirms that it is a member of the L-type family of Ca2+ channels.

What is the possible role of the two voltage-dependent Ca2+ currents in regulating heart beat in the snail Lymnaea?

We have identified and characterized four major currents in Lymnaea heart ventricle cells, two voltage-dependent K+ currents [IK(A) and IK(V)] (Yeoman and Benjamin 1999) and two voltage-gated Ca2+ currents (LVA and HVA) whose voltage sensitivities are shown in Fig. 9. Yeoman and Benjamin (1999) described the properties of small spikelike action potentials that could be evoked in dissociated heart muscle cells following hyperpolarization of cells from their resting membrane potential (-55 mV) to potentials around -63 mV (the lower operating range of the muscle cell, Fig. 9). Spikes were completely inhibited following superfusion of the cells with high concentrations (3.5 mM) of Cd2+. Based on this data and the voltage sensitivity of the Ca2+ currents obtained in this paper, we propose that the rising phase of ventricular action potentials is due to the sequential opening of LVA followed by HVA channels. The low activation threshold of the LVA current (-75 to -65 mV) and the relatively depolarized resting membrane potential of these cells (-55 mV) makes it ideally suited to provide the initial depolarizing drive to the cell. As mentioned in the previous paper (Yeoman and Benjamin 1999) the high-input resistance of these cells approx 900 MOmega means that a small current flow across the membrane will lead to a substantial depolarization of the cell. This depolarization would then activate the HVA current, which in turn would lead to a further depolarization of the cell (Fig. 9). Similar roles for T- and L-type calcium currents have been proposed for mammalian sinoatrial node cells (Hagiwara et al. 1988). The peak of the action potential (upper operating range of the muscle cell, Fig. 9) would occur when there was no net current flow across the muscle cell. This would be determined by the inactivation of the LVA and HVA currents and the relatively slow activation of IK(A) and IK(V). Further inactivation of the LVA and HVA currents and the continuing development of the K+ currents would lead to the repolarization of the muscle cell (Fig. 9). This is consistent with the observation that the falling phase of the action potential was sensitive to TEA, a K+ channel blocker (Yeoman and Benjamin 1999).



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Fig. 9. Schematic representation of the voltage sensitivities of the voltage-gated K+ and Ca2+ currents and their relationship to the proposed voltage operating range of Lymnaea heart ventricle cells. The operating range was defined at the lower end as the potential (-63 mV) that allowed spontaneous action potential generation and the upper level by the most positive voltage reached at the peak of the action potential (-11 mV).

The activity of the LVA current is typically seen as a fast activating, rapidly decaying current near -55 mV, the resting membrane potential of the cell. However, at more negative potentials (-70 to -60 mV), evoked currents are long-lasting and might therefore be able to provide a sustained depolarizing drive that could underlie repetitive spiking in these muscle cells as well as initiating individual beats. This observation may help explain why we found it necessary to hyperpolarize dissociated muscle cells from their resting membrane potential (-55 mV) to potentials near -63 mV, to obtain repetitive spiking in current-clamp experiments (Yeoman and Benjamin 1999). Hyperpolarization would also make more of the LVA current available for activation as well as helping to inactivate any residual K+ currents [IK(V)] thus causing the net current flow to become inward (pacemaker current). It was possible that by hyperpolarizing the muscle cells we were activating a different type of current such as Ih/If (hyperpolarization-activated current) that has been shown to drive pacemaker activity in the sinoatrial node (Difrancesco 1986). However, in a detailed analysis of >20 cells, we failed to show the presence of any hyperpolarization-activated currents. Thus in the absence of any other current, it appeared that the low-threshold voltage for the LVA channel made it a suitable candidate for providing the main current flow necessary for driving repetitive action potentials in these ventricle cells. Indeed, selective blocking of the LVA current by Ni2+ has been shown to cause a marked slowing of repetitive action potentials in rabbit sinoatrial node cells, indicating that this current does have a significant role in pacemaking in vertebrate heart cells (Hagiwara et al. 1988). Similarly, T-type currents have also been shown to underlie pacemaking and rhythmic activity in a number of neuronal structures such as the thalamic reticular nucleus and ventrobasal relay neurons of vertebrates (Huguenard and Prince 1994) and invertebrates (Adams and Benson 1985). We are currently performing similar experiments in snail muscle cells to determine the role of the LVA current.

Our observation that sustained LVA currents could occur at relatively depolarized potentials (-50 mV) in a few cells was an interesting one. Previous work has shown that LVA currents present in neurons have extremely heterogeneous kinetics. LVA currents have been typically classified into two classes according to their rates of inactivation. Thus currents are either fast inactivating transient currents or slow inactivating sustained currents (Adams and Levitan 1985; Eckert and Lux 1975). A number of these sustained LVA currents have been shown to be sensitive to DHP blockers and show a marked selectivity for Ba2+ over Ca2+ (Eckert and Lux 1976), suggesting that they are atypical L-type channels. In dissociated Lymnaea ventricle cells their appearance was extremely infrequent and as such precluded a study of their pharmacology and ion selectivity. However, their presence is interesting because they may contribute significantly to the pacemaker potential in the cells where they occur.

How might the voltage-gated currents present in Lymnaea ventricle cells contribute to action potential generation?

In a myogenic tissue such as the Lymnaea heart, action potential generation is of fundamental importance because it provides a mechanism that allows each muscle fiber to depolarize in the absence of neuronal input. In all excitable cells, whether an action potential is generated or not is determined by the balance of inward and outward currents flowing across the cell membrane. In some molluscan and vertebrate smooth muscles the ability of the cells to fire action potentials is prevented by the presence of large fast outward currents whose voltage sensitivity and kinetics are such that they are capable of counteracting the depolarizing drive due to the cationic inward currents, thereby blocking action potential generation. We have demonstrated that all dissociated ventricular muscle cells can generate action potential-like events, whose properties were a hybrid of those recorded from pacemaker cells in different regions of the mammalian heart (Irisawa et al. 1993). Two observations probably underlie our ability to record spikelike action potentials. The first is the relatively negative activation threshold of the LVA (-72 mV) currents compared with those for the two voltage-dependent K+ currents (Yeoman and Benjamin 1999). This would allow a significant depolarization (fast spike) via the influx of Ca2+ before it could be checked by a significant activation of the K+ currents (Fig. 9). The second is the faster activation rates of the Ca2+ currents compared with the two K+ currents that would also favor spiking.

Role of extracellular Ca2+ in excitation-contraction coupling?

In experiments described in Yeoman and Benjamin (1999) we determined that the threshold potential for activation of contractions in Lymnaea ventricle cells was -50 mV, with contractions increasing in strength as the potential of the cell was increased to -20 mV. Based on our recordings of ventricular action potentials, this probably represents the upper limit of the voltages reached by the cells under normal physiological conditions. Our observation that contractions start at potentials as negative as -50 mV is indicative that the first source of Ca2+ is via an influx through LVA Ca2+ channels, which are the only Ca2+ channels active at this potential (Fig. 9). Significant increases in intracellular Ca2+ levels due to influx via LVA channels have been described in a variety of different types of neurons (Markram and Sakmann 1994; Wang et al. 1997). However, recordable changes in intracellular Ca2+ levels due to influx via LVA channels in muscle are unusual with HVA channels providing the main entry pathway for this ion (Benham and Tsien 1988; Nelson et al. 1988). In the sinoatrial node the maximum currents that flow through T-type (LVA) channels are relatively small compared with those that flow through HVA channels (Hagiwara et al. 1988). In Lymnaea the situation appears to be different. Here the magnitude of the LVA current appears remarkably similar to that of the HVA current, although some caution should be taken when interpreting these data because it is not clear how the amplitude or the inactivation rate of the HVA current has been contaminated by the possible presence of unblocked K+ currents. Their presence would tend to reduce the peak amplitude and increase the apparent rate of inactivation. Despite this, we can see that there is a significant current flow through the LVA channels that would act to increase intracellular Ca2+ levels. Further depolarization of the muscle cell to potentials more positive than -50 mV would tend to activate HVA currents allowing these channels to contribute to the influx of Ca2+ (Fig. 9). The relative contributions of the LVA and HVA currents to elevating intracellular Ca2+ levels are at present unclear. However, current investigations involving selectively blocking of one or the other of the two Ca2+ currents should provide us with a clearer idea of the relative roles of these two currents.


    ACKNOWLEDGMENTS

M. S. Yeoman and P. R. Benjamin received financial support from United Kingdom Biotechnology and Biological Sciences Research Council Grant IR3521-1. B. L. Brezden was supported by a grant from the BBSRC Travelling Fellowship.


    FOOTNOTES

Address for reprint requests: M. S. Yeoman, School of Pharmacy and Biomolecular Sciences, Cockroft Building, University of Brighton, Moulsecoomb, Brighton, East Sussex BN2 4GJ, UK.

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. Section 1734 solely to indicate this fact.

Received 1 April 1999; accepted in final form July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society