ABSTRACT The accompanying paper (Josephson, I. R., A. Guia, E. G. Lakatta, and M. D. Stern. 2002. Biophys. J. 83:2575-2586) examined the effects of conditioning prepulses on the kinetics of unitary L-type Ca2+ channel currents using Ca2+ and Ba2+ ions to determine the ionic-dependence of gating mechanisms responsible for channel inactivation and facilitation. Here we demonstrate that in addition to alterations in gating kinetics, the conductance of single L-type Ca2+ channels was also dependent on the prior conditioning voltage and permeant ions. All recordings were made in the absence of any Ca2+ channel agonists. Strongly depolarizing prepulses produced an increased frequency of long-duration (mode 2) openings during the test voltage steps. Mode 2 openings also displayed >25% larger single channel current amplitude (at 0 mV) than briefer (but well-resolved) mode 1 openings. The conductance of mode 2 openings was 26 pS for 105 mM Ba2+ 18 pS for 5 mM Ba2+, and 6 pS for 5 mM Ca2+ ions; these values were 70% greater than the conductance of Ca2+ channel openings of all durations (mode 1 and mode 2). Thus, the prepulse-driven shift into mode 2 gating results in a longer-lived Ca2+ channel conformation that, in addition, displays altered permeation properties. These results, and those in the accompanying paper, support the hypothesis that multiple aspects of single L-type Ca2+ channel behavior (gating kinetics, modal transitions, and ion permeation) are interrelated and are modulated by the magnitude of the conditioning depolarization and the nature and concentration of the ions permeating the channel.
INTRODUCTION
Although it has been convenient to conceptualize ion channel gating as a simple two-state system (open and closed), most ligand-gated and voltage-gated ion channels display multiple conductance levels. For L-type Ca2+ channels, multiple conductance levels have been previously reported using cardiac myocytes (Chen and Hess, 1987), neurons (Church and Stanley, 1996), GH3 cells (Kunze and Ritchie, 1990), Ca2+ channel proteins reconstituted in bilayers (Ma and Coronado, 1988), and expressed al subunits of the L-type Ca2+ channel (Gondo et al., 1998; Cloues and Sather, 2000). However, the conditions that may promote a given conductance level remain largely unknown. Moreover, the properties of conductance states may contain important information concerning permeation and gating mechanisms necessary for a further understanding of the structure of the L-type Ca2+ channel.
High-voltage prepulses have been shown to facilitate single Ca2+ channel activity by promoting a mode of Ca2+ channel gating (one that is characterized by openings of unusually long duration, as compared with the briefer, mode 1 openings) using Ba2+ ions (Pietrobon and Hess, 1990; Hirano et al., 1999) or Ca2+ ions as the charge carrier (Josephson et al., 2002). These prepulse-facilitated single Ca2+ channel currents resemble the long-duration (mode 2) type of gating originally described for L-type Ca2+ channels during exposure to dihydropyridine agonists (Hess et al., 1984) or following /beta-adrenergic stimulation (Yue et al., 1990).
However, there is little information available concerning the conductance properties of mode 2 L-type Ca2+ currents, especially those recorded under more physiological conditions; that is, using a low concentration of Ca2+ ions as the charge carrier, and in the absence of L-type Ca2+ channel stimulation or agonists. In the present paper we focus on the conductance of unitary cardiac L-type Ca2+ channel currents displaying long-duration (mode 2) openings. We report that mode 2 L-type Ca2+ channel currents, recorded using Ca2+ and Ba2+ ions, are not only longer in duration, but also of greater conductance than briefer (but fully resolved) mode 1 openings. A preliminary report of some of these results has been presented in abstract form (Josephson et al., 2001c).
Increased conductance of mode 2 openings
To directly evaluate the conductance of mode 2 openings we measured the amplitudes of long-duration mode 2 openings that occurred near the end of a test pulse, and remained open during and after repolarization of the test pulse to the holding potential. The mode 2 single Ca 2+ channel "tail currents" had the advantage of yielding a slope conductance arising from individual, identifiable long-lasting openings of a single channel. As the deactivation of mode 2 is relatively slow, repolarization increases the electrochemical driving force on the permeating ions, and thus increases the single channel current amplitude. Examples of single mode 2 Ca2+ channel tail currents are shown in Fig. 3 A (traces a and b were recorded with 105 mM Ba2+ ions; c and d with 5 mM Ba2+ ions). Accompanying each current trace is the all-points histogram (Fig. 3 B) showing the amplitude of the long opening during the test pulse, and following repolarization. The current amplitudes were measured as the midpoints obtained from Gaussian fits to the all-points histograms.
The voltage-dependence for the amplitudes of single Ca2+ channel currents recorded during mode 2 tail current openings are compared with Ca2+ channel openings of all durations (recorded during single test steps, and identified and analyzed by a 50% threshold method) in Fig. 4. It should be noted that "all openings" included mode 2 openings as well as briefer mode 1 openings; however, the frequency of mode 2 events in the absence of a facilitating prepulse was usually <5% of the total number of openings. Part A displays results using 105 mM Ba2+ ions, part B with 5 mM Ba2+ ions. With 105 mM Ba2+ ions, the slope conductance was 25.7 +/- 1.2 pS for mode 2, whereas a linear regression to the average data for all openings (single steps) gave a slope conductance 14.5 +/- 0.5 pS. For 5 mM Ba2+ the slope conductance was 18.2 +/- 0.6 pS for mode 2, and was 10.8 +/- 0.4 pS for all openings. In addition to these differences in modal conductance, the apparent single channel reversal potentials were also different. For 105 mM Ba2+ ions, the extrapolated apparent reversal potential was +47 mV for mode 2 openings versus +65 mV for all openings; for 5 mM Ba2+ the extrapolated apparent reversal potential was +32 mV for mode 2 versus +38 mV for all openings.
As mode 2 tail current measurements were extremely rare with 5 mM Ca2+ ions (due to decreased frequency of reopenings near the end of the test step), mode 2 openings were measured during single voltage steps by the all-points amplitude histogram method, as shown in Fig. 5. Part A displays examples of mode 2 openings occurring during single voltage steps to the test potentials indicated. Part B shows the corresponding all-points histograms, constructed using segments of the traces surrounding the openings (as indicated by the arrows). The all-points histograms were fit with a sum of Gaussian functions to obtain the average amplitude of the mode 2 opening. With 5 mM Ca 2+ ions, a linear regression to the average amplitude data gave a slope conductance of 6.1 +/- 0.3 pS for mode 2, and 3.6 +/- 0.2 pS for openings of all durations (Fig. 5 Q. The extrapolated apparent reversal potential for 5 mM Ca2+ was + 32 mV for mode 2 versus +60 mV for openings of all durations.
Thus, with Ca2+ ions and Ba2+ ions, facilitation by high-voltage prepulses produced longer-duration mode 2 openings that attained a significantly greater conductance than shorter-duration (but fully resolved) openings. The implications of this novel voltage-dependent change in Ca2+ channel permeation (and gating kinetics) will be discussed subsequently.
DISCUSSION
The results of this paper and the accompanying paper (Josephson et al., 2002), demonstrate that multiple aspects of single L-type Ca2+ channel behavior (gating kinetics, modal transitions, and single channel conductance) are influenced by the magnitude of the conditioning depolarization and the nature and concentration of the permeant ion. A novel and important feature of the present results is the demonstration that strong depolarization not only resulted in a shift to mode 2 long-openings using a low concentration of Ca2+ ions, but also tended to temporarily "lock" the Ca2+ channel in its highest conductance conformation.
Similarly, an early report on L-type Ca2+ channels in smooth muscle cells (Caffrey et al., 1986) demonstrated that BayK8644, a Ca2+ channel agonist that pharmacologically promotes mode 2 long openings, also increased the single Ca2+ channel conductance by 25% (from 12 pS to 15 pS using 100 mM Ba2+ ions), and an increase in single channel current amplitude with CPG (another DHP derivative that also promotes mode 2) has been reported for cardiac L-type Ca2+ channels (Kokubun and Reuter, 1984). We have also found that another Ca2+ channel agonist, FPL 64176, increases the single channel conductance to the same level as that of mode 2 openings (Josephson, personal observation).
The conductance of single cardiac L-type Ca2+ channels has been reported over a wide range of values in previous studies, even in the absence of agonists and using the same divalent ion concentration (see Guia et al., 2001, for a review of the literature). In light of the present results, it seems plausible that contributing to at least a part of this range may be the variable number of mode 2 openings (as compared with mode 1) recorded in previous studies. The frequency of mode 2 openings (in the absence of voltagefacilitation or agonists) may be related to many factors, including species differences, endogenous intracellular levels of cyclic AMP or other second messengers, and the metabolic state of the myocytes. Moreover, mode 2 openings (that attain a stable amplitude level for an extended period of time) may have been favored in those previous studies where Ca2+ channel amplitude was measured by hand, thereby yielding a higher estimate of conductance.
In the present study mode 2 Ca2+ channel openings not only displayed a larger slope conductance but, in addition, the extrapolations of their slope conductances to the zero current level gave apparent reversal potentials that were less positive than those obtained from the conductance measurements obtained from all openings. This finding raises the intriguing possibility that during mode 2 the Ca 21 channel is temporarily less selective for divalent cations (i.e., Ca2+ and Ba2+), and that monovalent cations having a less positive reversal potential (such as cesium ions in our experiments, or sodium ions physiologically) may be allowed to permeate the channel. To speculate further, this loss of selectivity may be facilitated by high-voltage prepulses that might have the effect of driving divalent cations (i.e., Ca2+ ions) from their extracellular binding sites (perhaps in the pore region) that normally confer the divalent Ca2+ ion selectivity to the Ca2+ channel. Further experimentation using a variety of ionic conditions will be needed to test this novel hypothesis.
Physiological relevance
A voltage- and time-dependent switch promoting a mode 2 behavior of the Ca2+ channel would be a rapid and powerful mechanism to greatly enhance Ca2+ influx during an ongoing train of cardiac action potentials (activity-dependent potentiation). With a 10- to 100-fold increase in mean open time (Josephson et al., 2002) and a 70% increase in conductance, this facilitory mechanism may have a profound effect on the local control of excitation-contraction coupling (see Stem, 1992), whether in directly producing Ca2+induced Ca2+ release from the sarcoplasmic reticulum (SR) or in refilling the SR subsequent to Ca2+ release.
Even in the absence of activity-dependent potentiation, voltage-induced long openings might play an important role during individual action potentials. The plateau phase of the cardiac ventricular action potential (of most mammalian species besides rat and mouse) remains relatively constant for >100 ms at positive potentials. With a physiological Ca2+ ion concentration (1.8 mM) the voltage-dependence for activation of mode 2 would be shifted to less positive potentials, thus at plateau potentials long openings may be activated in a substantial number of Ca2+ channels. As the deactivation of mode 2 is relatively slow compared to the briefer mode 1 gating (especially at depolarized potentials), mode 2 openings would also be occurring during the repolarization phase of the action potential. In addition, as repolarization progresses the driving force for Ca2+ ion entry would also increase. The result of these factors would be a much larger influx of Ca2+ ion during the later phases of the action potential than would otherwise occur in the absence of this facilitory mechanism.
Facilitation of Ca2+ influx could also be potentiated by this voltage- and time-dependent mechanism in rat and mouse heart because in those species the high heart rate would activate mode 2 openings by summation over time, despite the very brief duration of each cardiac ventricular action potential. Thus, a late Ca2+ influx would occur during the repolarization phase of the action potential due to the relatively slow rate of deactivation of the mode 2 openings. Along the same lines, an enhanced Ca2+ influx produced by an augmentation of mode 2 activity during the abnormally rapid-firing, brief action potentials associated with ventricular fibrillation may contribute to Ca2+ overload and further myocardial damage. In addition, frequencydependent enhancement of mode 2 openings may have a role in modulating the activity of the sino-atrial nodal cells.
It also remains to be determined whether L-type Ca2+ channels that are capable of displaying this facilitory behavior may be anatomically localized (with respect to the Ca2+ release channels or other structures of the SR) to take functional advantage of this feature (e.g., in releasing Ca2+ ions, or in refilling the SR). Finally, although the most studied functions of the L-type Ca2+ current are in the electrogenesis of the cardiac action potential and in E-C coupling, we may also speculate that this voltage-dependent facilitation via mode 2 Ca2+ channel openings is involved in other Ca 2+-dependent signaling functions, such as activation of gene expression, or apoptosis.
In conclusion, the present results (which were obtained in the absence of any Ca2+ channel agonists and using a low concentration of Ca2+ ions and Ba2+ ions) demonstrate that strong depolarization drives the native cardiac L-type Ca2+ channel into a conformation that enables larger-amplitude, longer-duration openings. These findings suggest an intimate relationship of the voltage-sensing regions with the permeation-determining regions of the Ca2+ channel. It will be of great importance to gain a further understanding of the properties of these long openings as they undoubtedly play a important role in the local control of E-C coupling (Stem, 1992) in normal, aging, and diseased hearts.
The authors thank Bruce Ziman for excellent preparation of the isolated myocytes.
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[Author Affiliation]
Ira R. Josephson, Antonio Guia, Edward G. Lakatta, and Michael D. Stern
Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224 USA
[Author Affiliation]
Submitted February 27, 2002, and accepted for publication June 3, 2002.
[Author Affiliation]
Address reprint requests to Dr. Ira Josephson, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224. Tel.: 410-558-8644; Fax: 410-558-8150; E-mail: josephsoni@grc.nia.nih.gov.
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