FPL-64176 modifies pore properties of L-type Ca2+ channels

Jing-Song Fan1, Yuhui Yuan1, and Philip Palade1,2

Departments of 1 Physiology and Biophysics and 2 Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-0641


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In addition to its known effects on Ca2+ and Ba2+ currents, the L-type Ca2+ channel agonist FPL-64176 was found to affect channel function in isolated rat ventricular myocytes in the absence of Ca2+, with other ions as current carriers through the channel. FPL-64176 induced Cd2+ current through the L-type Ca2+ channel, suggesting that certain selectivity properties had changed, perhaps indicative of a small change in pore structure. FPL-64176 slightly but significantly decreased the effectiveness of Co2+ as a blocker of the channel. FPL-64176 also increased conductance through single L-type Ca2+ channels recorded in the cell-attached configuration, from 71.9 ± 11.6 to 94.1 ± 8.3 pS, with Na+ carrying the current at pH 9.0. At present it is uncertain whether FPL-64176 produces small alterations of a sole open state of the channel or whether it increases the prevalence of a second, higher conductance open state. These changes, particularly the conversion of Cd2+ from a pure blocker to a permeant ion, may be of eventual help in discriminating among different models for Ca2+ channel selectivity.

cardiac myocytes; Cd2+; Co2+; conductance; selectivity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CALCIUM CHANNEL AGONIST FPL-64176 has been used by several different groups in studies of cardiac excitation-contraction coupling because it reduces the rate of decay of L-type Ca2+ currents and enables identification of a separate adaptation or inactivation process that turns off Ca2+ release mediated by ryanodine receptors (28, 30). In the process of determining its effects on Ca2+ channel gating currents and the Ca2+ dependence of its actions on inactivation, we examined the effects of FPL-64176 on the channel with other ions carrying the current.

Most studies of FPL-64176 have concentrated on alterations in the time course of macroscopic or single-channel currents. By comparison, far less has been reported of effects on pore properties of the channel. One report of the effect on single L-type channels in failing human heart suggested that single-channel conductance might be increased (11). In the process of studying the effects of FPL-64176 on L-type gating currents (8), we determined that Cd2+, used by others for gating current measurements, is capable of carrying current through the channel in the presence of FPL-64176. Some of these results have been reported previously in abstract form (7).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myocyte dissociation. Rat ventricular myocytes were prepared from 200- to 300-g male Sprague-Dawley rats by dissociation with collagenase (Yakult Pharmaceuticals, Tokyo, Japan) as previously described (5, 6, 32). Myocytes were stored at 4°C in a high-potassium, low-sodium, Kraftbrühe-like solution (33) until use.

Measurement of current through L-type Ca2+ channels. Patch-clamp (List EPC-7) measurements were performed with a perforated-patch version of the whole cell recording configuration with the use of 50 µM beta -escin in the pipette solution (5) to circumvent Ca2+ channel rundown. The compositions of experimental external solutions utilized in this study are given in Tables 1 and 2. The internal (pipette) solution consisted of 120 mM Cs-aspartate supplemented with 20 mM CsCl, 3 mM Na2ATP, 3.5 mM MgCl2, 5 mM EGTA, and 5 mM HEPES, pH 7.3. Holding potential was -40 or -50 mV in most experiments on selectivity. Leak and capacity compensation was generally performed manually with the patch-clamp controls by using 10-mV hyperpolarizations from the holding potential. For the more critical experiments shown in Figs. 3 and 4, leak and linear capacitance corrections were carried out digitally by utilizing 25-mV hyperpolarizing pulses from a holding potential of -100 mV. All experiments were performed at room temperature. FPL-64176 and S(-)-BAY K 8644 were obtained from RBI (Natick, MA).

                              
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Table 1.   Composition of external solutions with principal current carriers


                              
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Table 2.   Composition of external solutions involving Cd2+ or Co2+

Single-channel measurements were performed in the cell-attached recording configuration by using a Dagan 3900 patch clamp. Normal Tyrode solution contained 1 mM Ca2+, with Cs+ substituted for K+ for purposes of blocking inward rectifier currents in most experiments. Under these conditions, the resting potential of the cells approximated -65 mV. Two pipette solutions were used. The first consisted of Ca2+- and Cs+-free Tyrode solution with pH adjusted to 9.0 (12). The second consisted of 70 mM BaCl2 and 110 mM sucrose, pH 7.4 (3, 11). FPL-64176 was added to the bath solution in all cases. All experiments were performed at room temperature with filtering at 1 kHz and sampling every 0.5 ms. Analysis of records was performed by using pCLAMP 6, with cursors set at 50% of the full current openings for kinetic analysis.

Statistical analysis. Statistical significance was assigned at the P < 0.05 level, using the unpaired Student t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of FPL-64176 are apparent with other ions carrying current through the channel. In some cases the effects of the standard 1 µM FPL-64176 employed in L-type Ca2+ current studies (8) were so marked that lower FPL-64176 concentrations needed to be employed to avoid inducing unacceptably large increases in holding current. As shown in Fig. 1 and Table 3, Sr2+, Ba2+, and Na+ currents through the L-type channel all were affected by FPL-64176. The traces in Fig. 1 reveal that channel activation continued for a longer time than normal, resulting in less decay of the current during the pulse. Furthermore, tail currents were enhanced, and a portion of the tail exhibited a very slow decay. This finding is reflected in the analysis provided in Table 3, where it is shown that the vast majority of the tail current declined with a fast time constant of ~2 ms in control records in each ion, but both fast and slow time constants were significantly increased in the presence of FPL-64176, and the proportion of the tail exhibiting the slow time constant, now in excess of 100 ms, was also increased.


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Fig. 1.   FPL-64176 affects the channel if Sr2+, Ba2+, or Na+ carries current through the L-type channel instead of Ca2+. Top: currents carried by 1 mM Sr2+ substituted for Ca2+ in the extracellular solution under control conditions (left) and in the presence of 0.1 µM FPL-64176 (right). Middle: currents carried by 1 mM Ba2+ substituted for Ca2+ in the external solution under control conditions and in the presence of 0.1 µM FPL-64176. Bottom: currents carried by Na+ in the extracellular solution in the absence of Ca2+ (with 0.5 mM EGTA added to nominally Ca2+-free external solution) under control conditions and in the presence of 0.1 µM FPL-64176. All currents were measured in response to stimulations to 0 mV from a holding potential of -40 mV. Results are representative of 3 experiments each with Ba2+ or Sr2+ as current carrier and 2 experiments with Na+ as current carrier.


                              
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Table 3.   Effects of FPL-64176 on currents carried by Sr2+, Ba2+, or Na+

Peak currents in Ba2+ remained slightly larger than those in Ca2+ solutions, as evidenced by the traces shown in Fig. 2, left. However, the difference is not statistically significant. The current-voltage relationships for currents carried by the two charge carriers were different in the presence of FPL-64176 (Fig. 2, right). The shifts observed in the presence of Ba2+ were accompanied by a small but significant increase in inward holding current, and the reversal potential for current flow through the channel was shifted in the negative direction, consistent with a possible buildup of intracellular Ba2+. With these observations taken into consideration, there do not appear to be major changes in the relative permeabilities of Ba2+ and Ca2+ through the channel.


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Fig. 2.   Ba2+ continues to pass slightly more current than Ca2+ through FPL-64176-modified channels. Left: traces of currents carried by 1 mM Ca2+ (top) and 1 mM Ba2+ (bottom) in response to depolarizations from -30 to +80 mV from a holding potential of -40 mV are shown in ventricular myocytes exposed to 0.1 µM FPL-64176. Right: current-voltage plots of the relationships in 4 such FPL-64176-treated cells each successively in 1 mM Ca2+ and 1 mM Ba2+ solutions. Vt, test potential.

FPL-64176 renders Cd2+ permeable through the channel. Unlike the highly permeant ions Sr2+, Ba2+, and Na+, Co2+ and Cd2+ are normally impermeant through the channel. As shown in Fig. 3, the combinations of either Co2+ and triethanolamine (Tri) or Co2+ and tetraethylammonium (TEA) yielded no increase in current in the presence of FPL-64176. TEA solution without divalents also failed to pass inward tail current in the presence of FPL-64176 (not shown). Thus neither Co2+, Tri, nor TEA is permeant in the presence of FPL-64176. In contrast, Cd2+/Tri and Cd2+/TEA carried inward current through the channel. The effects were most apparent during the tails that followed repolarization. The enhanced tail currents suggest that Cd2+ is permeant through the FPL-64176-modified channel.


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Fig. 3.   Altered channel selectivity in the presence of FPL-64176. Tail currents in response to repolarization from +10 to -50 mV are shown in the presence of only normally impermeant cations in the extracellular solution. Top to bottom: Co2+ + triethanolamine (Tri) (n = 3); Co2+ + tetraethylammonium (TEA) (n = 4); Cd2+ + Tri (n = 3); and Cd2+ + TEA (n = 3). Numbers in parentheses indicate no. of representative experiments performed. Left: control conditions; right: corresponding records in the same solutions in the presence of 1 µM FPL-64176. Note that there was no FPL-64176-induced increase in inward current in Co2+-containing solutions, but current clearly increased in solutions containing Cd2+.

Further Cd2+ tail current measurements are shown in Fig. 4, left, where the pulse protocol involved pulses to different potentials, followed by a return to the -50-mV holding potential. Under control conditions, essentially only gating currents were observed. In the presence of FPL-64176, significant inward current was observed during the pulse, but much larger tail currents were observed during repolarization. In Fig. 4, right, peak inward tail currents are plotted, with approximate compensation for gating currents achieved by subtraction of the corresponding "on" gating current from the tail. In the absence of FPL-64176, entry of Cd2+ was clearly insignificant, whereas in the presence of FPL-64176, the current reached very large amplitudes. As with Ca2+ current in the presence of FPL-64176 when the same protocol was used (not shown), Cd2+ tail currents continued to increase as the prepulse potential was made quite positive.


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Fig. 4.   Cd2+ tail current in response to depolarizations to different potentials. Left: current traces in Cd2+ solution in response to depolarizations from -30 to +40 mV from a holding potential of -50 mV under control conditions (top) and in the presence of 1 µM FPL-64176 (bottom). Right: current-voltage plot of instantaneous tail current (Itail) elicited on repolarization to -50 mV, with "on" gating current amplitudes subtracted from the "off" tail responses. Leak and capacity compensation was performed by subtracting scaled responses to 25-mV hyperpolarizations from a holding potential of -100 mV. Results are pooled from 3 cells in Cd2+-Tri solution and 3 cells in Cd2+-TEA solution (for compositions, see Table 1) before and after treatment with FPL-64176. *P < 0.05.

Additional experiments were conducted to examine possible channel rectification of Cd2+ current in the presence of FPL-64176. A prepulse to near the normal Ca2+ equilibrium potential was applied for a period long enough to open most channels. Subsequently, the membrane was repolarized to different potentials to elicit Ca2+, Cd2+, or Co2+ inward currents. Traces in the presence of Co2+ were then subtracted from those in the presence of Ca2+ or Cd2+, resulting in the families of traces shown in Fig. 5, left. Currents carried by 3 mM Cd2+ were half the size of those observed in the presence of 1 mM Ca2+, in the presence of FPL-64176. In Fig. 5, top right, it may be seen that there was no equivalent detectable Cd2+ current in the absence of FPL-64176. There is rectification of the curve in the presence of Ca2+ because the tail currents became very brief and attenuated at potentials below -20 mV. In contrast, in the presence of FPL-64176, this attenuation was shifted to more negative potentials (not shown) such that the driving force increases nearly linearly with voltage over the range from +20 to -40 mV, for both Cd2+ and Ca2+ (Fig. 5, bottom right).


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Fig. 5.   Cd2+ current through the channel in the presence (+) of FPL-64176 is significant even compared with Ca2+ current. Left: current traces in response to a strong depolarization to +80 mV, followed by repolarization to different potentials from +70 (smallest tails) to -40 mV (largest, most swiftly declining tails). Individual cells were exposed to Ca2+/TEA (top) or Cd2+/TEA solution (bottom), followed by Co2+/TEA solution. Traces in Co2+/TEA were subtracted from traces in other solutions to provide as much leak and capacity compensation as possible. Top right: current voltage plots of traces under control conditions, in the absence (-) of FPL-64176, demonstrating an absence of Cd2+ tail current (n = 3). Bottom right: current-voltage plot of traces in the presence of 1 µM FPL-64176 (n = 4). Vr, repolarization potential.

FPL-64176 reduced the effectiveness of Co2+ as a blocker. Figure 6 demonstrates that the efficacy of Co2+ as a blocker was reduced in the presence of FPL-64176. In this case, Ca2+ was the current carrier. In these experiments, progressively higher concentrations of Co2+ were added to the external solution. As shown in Fig. 6, top, there was generally even a small increase in current (run-up) at the beginning of the experiments, perhaps because of slow enhancement caused by the FPL added earlier. At higher concentrations, blockers always inhibited the current, and the effects were quite reversible. Plots in Fig. 6, bottom, show the results from several such experiments with Co2+.


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Fig. 6.   Co2+ is a less effective blocker in the presence of FPL-64176. Top: Ca2+ currents (ICa) in the presence of 1 µM FPL-64176 were progressively blocked by increasing concentrations of Co2+ added cumulatively. Bottom: block by Co2+ of normalized ICa elicited under control conditions (n = 5) or in the presence of 1 µM FPL-64176 (n = 4), with Co2+ added cumulatively to the bathing solution. *P < 0.05 compared with control.

FPL-64176 affects single-channel conductance. We also wanted to determine whether single-channel conductance was increased by FPL-64176. In the presence of 1 µM FPL-64176 in 70 mM BaCl2 and 110 mM sucrose, three cells gave values of 28.2, 23.2, and 27.1 pS (not shown). We encountered difficulty in identifying Ca2+ channel openings and measuring current amplitudes in the absence of drug, but conductance in the presence of drug was not significantly different from that reported in rat ventricular myocytes in the same solution without FPL-64176 (27.7 ± 0.7 pS; Ref. 3).

To pursue this matter further, we employed other solutions. Recordings of single L-type Ca2+ channel activity in the absence of Ca2+ channel agonist were facilitated by experimental conditions that yielded large-amplitude currents (monovalent Na+ passing through the channel at pH 9.0; Refs. 21-23). A typical determination is shown in Fig. 7, and all determinations included data points at four or more potentials. In the absence of agonist, the conductance of the channel with current carried by Na+ at pH 9.0 was 77.4 ± 18.2 pS (n = 8). In the presence of 1 µM FPL-64176, openings were resolved over a wider range of potentials, including responses obtained on repolarization to the resting potential. The conductance measured under the same conditions in the presence of FPL-64176 was 94.1 ± 8.3 pS (n = 9, P = 0.025 vs. control; i.e., significantly different statistically). We found only occasional long openings in the absence of FPL-64176. Because short openings in the controls might have resulted in underestimates of the true conductance, we were uncertain whether to utilize long opening data under control conditions. The above analysis included two such long openings. When these two long openings were discarded from the analysis, the control conductance was decreased, the data became less variable (73.3 ± 12.2 pS, n = 8), and the statistical significance of the differences between control and FPL-64176 data improved to P = 0.00086. 


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Fig. 7.   Single-channel activity due to L-type Ca2+ channels and its modification by FPL-64176 in Na+ solution, pH 9.0. Top: single sweeps of activity are shown for depolarizations of 10 and 20 mV from the estimated resting potential of -65 mV under control conditions (left) and in the presence of 1 µM FPL-64176 (right). Note the increased open probability both during and after the 20-mV stimulus. Records were obtained in control Na+ solution, pH 9.0, from a patch with 2 channels present. In certain cases the background subtraction of leak and capacity was imperfect. All channel openings are downward. Bottom: FPL-64176 increased the single-channel conductance (gamma ) of L-type Ca2+ channels. Records such as those shown at top were used to determine the single-channel conductance. Potentials indicate excursions from the resting potential.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our characterization of effects of FPL-64176 on the pore properties of the L-type Ca2+ channel includes changes in which ions can pass through the channel (a measure of selectivity), single-channel conductance, and block by ions thought to interact with binding sites in the pore.

Selectivity. FPL-64176 exerted its characteristic effects with ions other than Ca2+ carrying the current. Not only were the currents carried by divalents like Ba2+ and Sr2+ affected, but so were currents carried by Na+. In our limited experiments with these ions carrying the current, it did not appear that currents carried by one of these ions were increased much more than currents carried by the others. Thus, to a first approximation, FPL-64176 did not seem to affect the ability of the channel to discriminate among the principal known current carriers through the channel.

Because FPL-64176 exerted more dramatic effects on amplitude of Cd2+ currents compared with those carried by Ca2+ or Ba2+, effects of the drug on currents carried by other divalents were also examined (with TEA as the other extracellular cation). Zn2+ was found to carry marginal current through the channel in the absence of drug and much greater current in the presence of drug (n = 4, not shown). In contrast, Mg2+ (n = 2), Ni2+ (n = 3), and Be2+ (n = 2) carried no current through the channel in either the absence of drug or the presence of FPL-64176 (not shown). While most permeant divalents have larger crystal radii than blockers like Cd2+, the ability of the even smaller Mn2+ (1, 20) and Zn2+ ions to pass through the channel normally suggests that crystal radius is not an important determinant of selectivity. Similar arguments suggest that hydrated ion size also is not a determinant of selectivity and that FPL-64176 is not simply affecting the diameter of the pore. We speculate instead that the affinity of Cd2+ for its high-affinity site in the pore (29) could be reduced to the point that it becomes more susceptible to being knocked through the channel by the presence of a second Cd2+ ion in the pore (as for Ca2+ in Refs. 13 and 18). Alternatively, either its high-affinity site or the postulated innermost lower affinity site in the "step" model of Dang and McCleskey (4) could be altered. In principle, speculation about alteration of the higher affinity site could be tested by performing equivalent Cd2+ block experiments, as performed with Co2+ and shown in Fig. 6. Unfortunately, Cd2+ block was much slower, progressive, and poorly reversible than Co2+ block (not shown), and this rendered equivalent assessment of IC50 for Cd2+ unfeasible.

These experiments also indicated that FPL-64176 did not require Ca2+-bound states of the channel to exert its effects. Replacement of Ca2+ in our solutions would have diminished its influence, and while Sr2+ has been reported to be capable of releasing Ca2+ from the sarcoplasmic reticulum (25), Ba2+ and Na+ would not (19). The finding that similar FPL-64176-induced kinetic changes occurred with other ions substituting for Ca2+ as a current carrier argues against an obligatory role for Ca2+ in mediating the effects of FPL-64176. Consequently, even though certain models of the L-type channel include a Ca2+-dependent inactivation or Ca2+-bound mode of the channel (14), such states must not be required for FPL-64176 to exert its effects.

The observation that Cd2+ passes through FPL-64176-modified channels but not through untreated channels was unexpected and is entirely novel. Although this occurrence initially hindered experiments designed to measure the effects of the drug on L-type Ca2+ channel gating currents, it demonstrates that the pore properties of the channel are changed by the drug. Either the pore region of the channel is made slightly wider or the affinity of Cd2+ to a binding site within the pore is affected. Of these two possibilities, we favor the latter because TEA, a marginal current carrier through skeletal L-type channels (18), remained impermeant even in the absence of divalent blockers (not shown). Cd2+ is a well-known L-type channel blocker whose effects are voltage dependent (16), indicating that it binds to a site within the permeation pathway. Significantly, Cd2+ has been reported to be permeable through insect muscle Ca2+ channels (9). In addition, all divalent current carriers through this channel are also known to block movement of monovalents through the channel (13). Thus the conversion of Cd2+ from blocker to current carrier should not be so surprising. Other drugs have been reported to induce changes in selectivity of other cardiac channels (26).

Because most experiments assessing effects of BAY K 8644 on mammalian myocyte Ca2+ channel gating currents have been carried out with Cd2+ present (10), we also tested whether BAY K 8644 caused an increase in current carried by Cd2+. No increases in tail currents were noted when Co2+ was present (n = 8, not shown). However, inward currents were clearly apparent during the pulse, and tails were slowed in Cd2+/Tri (n = 2, not shown) and slowed and increased in Cd2+/TEA (n = 3, not shown). Thus it appears that BAY K 8644 may increase channel permeability to Cd2+, although not as much as FPL-64176.

Conductance. Another potential pore property is the single-channel conductance. Handrock et al. (11) recently reported that FPL-64176 increased single-channel conductance with Ba2+ as a charge carrier in human ventricle. We tested for an increase with Na+ as the charge carrier instead.

Large-amplitude currents carried by Na+ through L-type Ca2+ channels were first reported by the late Peter Hess and colleagues (12). Still larger amplitude events were obtained with alkaline pH patch-pipette solutions (21-23). Na+ also normally carries current through sodium channels, and it can also carry current through T-type channels in cardiac myocytes in the absence of divalents. Nevertheless, Na+ single-channel conductance is much lower (2) than that shown here, even at alkaline pH (31, 34), and channel activity dies off much faster (27) than during the observations made here. T-type Ca2+ currents are much smaller than L-type currents in adult myocytes, and single-channel conductances in T-type channels conducting Na+ are much lower than those of L-type channels, even at alkaline pH (15). Thus we can be confident that our recordings were from L-type Ca2+ channels.

The values we obtained under control conditions are generally less than those obtained by Hess and coworkers (12) using another Ca2+ channel agonist, BAY K 8644 (85 pS) (12), but our recordings in the presence of FPL-64176 are generally greater than those. Recordings in similar solutions with another agonist, (+)-S-202-791, appear very similar (22) or were not quantified in terms of single-channel conductance (23, 24). The increase in single-channel conductance that we observed with FPL-64176 with Na+ as a charge carrier is nevertheless in agreement with the increase from 16.6 ± 1.2 to 23.7 ± 2.8 pS observed in human ventricular myocytes in Ba2+ solution (11).

Three possibilities could be contemplated to explain the modest increase in single-channel conductance that we observed in the presence of FPL-64176. The first possibility is that short openings in our control records caused us to selectively underestimate the true single-channel conductance in the absence of drug. We regard this as relatively unlikely because we always selected the longest openings for these determinations, and the histograms from these experiments indicate mean openings of ~2 ms in the absence of drug with 1-kHz filtering (8). The second possibility is that the two gating modes observed in the absence of drug (12, 21) actually have two different single-channel conductances and that FPL-64176 enhances mode 2-type gating. An argument in favor of such an interpretation is that conductance measurements that included mode 2-like long openings on repolarization appeared to yield higher conductance estimates than those that excluded such openings. An argument against such an interpretation is that Hess et al. (12) observed no difference in unitary current between control and BAY K 8644-treated patches. Furthermore, they found no difference in amplitudes between mode 1 and mode 2 openings. The third possibility is that FPL-64176 causes the channel to be modified into a higher conductance state not found under control conditions. An argument in favor of this interpretation is the finding that Cd2+ was rendered permeant by FPL-64176.

Modification of channel block. The block of the permeation pathway by Cd2+ was affected in a striking fashion, converting it from a blocker of the channel to a current carrier. While this conversion might be unique to Cd2+, the blocking action of Co2+ was also affected by FPL-64176, albeit in a more subtle fashion. In previous work, we used 3-4 mM Co2+ to block Ca2+ movement through the channel to separate ionic current from capacitative current (6). In the experiments reported here, the effective IC50 for Co2+ was shifted from 0.50 ± 0.26 to 1.62 ± 0.73 mM (P < 0.05) in the presence of FPL-64176.

Overall effects on pore properties. In summary, FPL-64176 affects L-type Ca2+ channels even with ions other than Ca2+ carrying the current. It renders Cd2+ permeant and reduces the effectiveness of Co2+ as a blocker of the channel pore. Finally, it either favors an open state of the channel with higher conductance than the normal principal open state or actually causes the channel to open to a higher conductance state than normal. The modification of Ca2+ channel selectivity by FPL-64176 might prove useful in discriminating among different models for Ca2+ channel permeation (17).


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants AR-41526 and AR-43200 (to P. Palade).


    FOOTNOTES

Present address of Y. Yuan: Dept. of Cell Biology, Baylor College of Medicine, Houston, TX 77030.

Address for reprint requests and other correspondence: P. Palade, Dept. of Physiology and Biophysics, Univ. of Texas Medical Branch, Galveston, TX 77555-0641 (E-mail: ppalade{at}utmb.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. Section 1734 solely to indicate this fact.

Received 28 March 2000; accepted in final form 20 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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