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The Journal of Neuroscience, February 15, 2001, 21(4):1137-1147
G-Protein Inhibition of N- and P/Q-Type Calcium Channels: Distinctive Elementary Mechanisms and Their Functional Impact
Henry M. Colecraft, David L. Brody, and David T. Yue Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Voltage-dependent G-protein inhibition of presynaptic Ca2+ channels is a key mechanism for regulating synaptic efficacy. G-protein
subunits produce such inhibition by binding to and shifting channel opening patterns from high to low open probability regimes, known respectively as "willing" and "reluctant" modes of gating. Recent macroscopic electrophysiological data hint that only N-type, but not P/Q-type channels can open in the reluctant mode, a distinction that could enrich the dimensions of synaptic modulation arising from channel inhibition. Here, using high-resolution single-channel recording of recombinant channels, we directly distinguished this core contrast in the prevalence of reluctant openings. Single, inhibited N-type channels manifested relatively infrequent openings of submillisecond duration (reluctant openings), which differed sharply from the high-frequency, millisecond gating events characteristic of uninhibited channels. By contrast, inhibited P/Q-type channels were electrically silent at the single-channel level. The functional impact of the differing inhibitory mechanisms was revealed in macroscopic Ca2+ currents evoked with neuronal action potential waveforms (APWs). Fitting with a change in the manner of opening, inhibition of such N-type currents produced both decreased current amplitude and temporally advanced waveform, effects that would not only reduce synaptic efficacy, but also influence the timing of synaptic transmission. On the other hand, inhibition of P/Q-type currents evoked by APWs showed diminished amplitude without shape alteration, as expected from a simple reduction in the number of functional channels. Variable expression of N- and P/Q-type channels at spatially distinct synapses therefore offers the potential for custom regulation of both synaptic efficacy and synchrony, by G-protein inhibition.
Key words:
1A;
INTRODUCTION
Voltage-dependent G-protein inhibition of N- and P/Q-type channels is a ubiquitous and potent mechanism for presynaptic inhibition of neurotransmitter release (Lipscombe et al., 1989
; Wu and Saggau, 1994
) to N- and P/Q-type channels (Herlitze et al., 1996
Whether G
We have recently suggested, based on indirect whole-cell recordings, that N-type, but not P/Q-type channels can open in the reluctant mode (Colecraft et al., 2000
Here, we report the single-channel mechanisms underlying voltage-dependent G-protein inhibition of recombinant N- and P/Q-type channels, as reconstituted in human embryonic kidney (HEK 293) cells. This system permits unambiguous identification of channel type and reconstitution of a purely voltage-dependent G-protein inhibitory mechanism (Patil et al., 1996
MATERIALS AND METHODS
Transfection of HEK 293 cells. HEK 293 cells were maintained as previously described (Brody et al., 1997
2a (Perez-Reyes et al., 1992
(Tomlinson et al., 1993
Electrophysiological recordings. Both whole-cell and single-channel currents were recorded at room temperature, 2-3 d after transfection, using an Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster City, CA). For whole-cell recordings, cells were continuously perfused with a bath solution containing in mM: 150 tetraethylammonium methanesulfonate (TEA-MeSO3), 2 CaCl2, 1 MgCl2, and 10 HEPES, adjusted to pH 7.4 with TEA-OH. Where noted, 50 µM carbachol was added to the bathing medium to activate G-proteins via the cotransfected m2 receptor. The internal solution contained (in mM): 135 cesium methanesulfonate (Cs-MeSO3), 5 CsCl, 10 EGTA, 1 MgCl2, 10 HEPES, and freshly added 4 MgATP, pH 7.3, adjusted with CsOH. Electrodes were fashioned from borosilicate glass capillaries (WPI MTW 150-F4) and had pipette resistances ranging from 1.2 to 2.5 M
, before series resistance compensation of 75%. Voltage pulses were applied at 30 sec intervals. Currents were filtered at 5 kHz for action potential (AP) trains, or at 10 kHz for tail current measurements (four-pole Bessel), and digitized at 25 or 50 kHz, respectively. Voltages were corrected for a liquid junction potential of
11 mV before recording, and leak and capacitance transients subtracted by a P/8 protocol. AP voltage templates were based on recordings from the Calyx of Held (Borst et al., 1995
Single-channel and macropatch currents were obtained in the cell-attached patch-clamp configuration. To zero the membrane potential, the bath contained a solution consisting of (in mM): 132 K-glutamate, 5 KCl, 5 NaCl, 3 MgCl2, 2 EGTA, 10 glucose, and 10 HEPES, pH 7.4, adjusted with KOH. The pipette solution contained 90 mM BaCl2, 20 mM TEA-MeSO3, 50 µM carbachol (where indicated), and 10 mM HEPES, pH 7.4, adjusted with TEA-OH. Patch pipettes were fashioned from fused quartz capillaries (Garner Glass Company, Claremont, CA) using a laser puller (Sutter Instrument, Novato, CA) and coated with Sylgard. Pipettes typically had resistances ranging from 5 to 20 M
Single-channel data analysis. All patches analyzed in this study contained a single active channel. Custom analysis software, written in Matlab, was used throughout. Records were additionally filtered to a 1.8 kHz bandwidth using a digital Gaussian filter and idealized using half-height criteria. Idealized records were used for generation of histograms, ensemble averages, and Poo distributions. Ensemble average currents, first-latency distribution (FL), and conditional open probability (Poo) were calculated as previously described (Imredy and Yue, 1994
Statistics. Throughout, pooled data are presented as mean ± SEM. All p values were calculated from two-tailed t-test; p < 0.05 was considered as significant.
RESULTS
Large surface-potential shifts complicate physiological interpretation of single-channel data
To facilitate resolution of single, Ca2+-channel gating events with adequate frequency bandwidth, pipette solutions typically contain
90 mM Ba2+ to increase unitary current amplitude. Such high concentrations of divalent cations screen negative surface charges much more effectively than would physiological concentrations of Ca2+ (Frankenhaeuser and Hodgkin, 1957
High-level expression of recombinant N-type (
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Figure 1. Magnitude of surface potential shift in cell-attached recordings of calcium channel activity, with 90 mM Ba2+ as charge carrier. A, Top, Voltage protocol. Bottom, Exemplar N-type channel whole-cell tail current evoked after a step depolarization to +10 mV, with 2 mM Ca2+ as charge carrier. B, Top, Voltage protocol. Bottom, Exemplar N-type channel macropatch tail current obtained in a cell-attached recording, with 90 mM Ba2+ as charge carrier. Displayed tail current was evoked after a step depolarization to +50 mV. C, Comparison of N-type channel whole-cell (open circles) or cell-attached macropatch (filled circles) tail activation (G-V) curves. Smooth curves through the data are least-squares single Boltzmann fits with the following parameters: whole-cell (V1/2,
Single-channel gating properties of uninhibited N-type channels
The surprisingly large surface-potential shift of voltage-dependent gating under single-channel recording conditions challenged us to undertake considerably stronger single-channel depolarizations than previously attempted, to establish gating properties with greater relevance for physiological behavior, and to test appropriately for unitary reluctant openings. Such stronger depolarization would incur substantial drops in electrochemical driving force with resulting diminution of unitary current amplitudes. Nonetheless, stronger depolarization was an absolute requirement here, given that our traditional single-channel experiments (Patil et al., 1996
As a prelude to G-protein modulation experiments, Figure 2 illustrates good resolution of the baseline characteristics of single, unmodified N-type channels, despite test-pulse depolarization to +45 mV. Quartz pipettes proved invaluable, here and throughout, in reducing noise sufficiently to distinguish elementary events during strong depolarization (Levis and Rae, 1998
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Figure 2. Baseline single-channel gating characteristics of recombinant N-type channels. A, Top, Voltage protocol. Test pulses without (left) or with (right) a prepulse were interleaved. Bottom, Representative unitary current activity from a patch containing a single N-type channel. Here and throughout, representative records are not consecutive, and dotted lines represent the zero current level, unless otherwise stated. The mean unitary current amplitude was 0.65 ± 0.05 pA (n = 3). B, Ensemble average currents averaged from three patches. Here and throughout, ensemble currents were averaged from all sweeps, including nulls. C, FL distributions obtained either without (gray trace) or with (black trace) a prepulse. D, Conditional open probability distributions, Poo, obtained without (gray trace) or with (black trace) a prepulse. Smooth curve is a least-squares fit of a biexponential function to the no prepulse data. E, Open time distribution histograms of N-type channels obtained either without (left), or with (right) a prepulse. The number of openings with duration more than or equal to the time interval on the x-axis was normalized by the total number of openings estimated from maximum likelihood fits [yielding # events/bin (norm.)], and plotted on log-log coordinates with bin width fixed at 40 µsec throughout. Maximum likelihood fits to the data had the following parameters: 1 = 0.61 msec, 66%;
Beyond demonstrating the feasibility of acquiring single-channel data at strongly depolarized test potentials, these results also provided immediate dividends by permitting the first calibration of macroscopic tail-activation curves (Fig. 1) in terms of absolute open probabilities Po, a long-sought-after feat with important implications for the physiological performance of presynaptic Ca2+ channels (Borst and Helmchen, 1998
Single-channel detection of reluctant N-type channel openings during G-protein inhibition
We next turned to single-channel investigation of N-type channels during G-protein inhibition, using strong depolarizations as might be required to elicit reluctant openings (Fig. 3). The protocols and display format were identical to those used in the controls (Fig. 2), except that G-protein activation was produced by the inclusion of saturating 50 µM carbachol (CCh) in the patch pipette to activate coexpressed m2 muscarinic receptors (Patil et al., 1996
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Figure 3. With depolarizations to +45 mV, G-protein-inhibited single N-type channels display slowly activating reluctant openings. A-F, Identical format as in Figure 2. A, Mean unitary current amplitude was 0.62 ± 0.03 pA (n = 3). B, Ensemble average currents. Smooth gray traces overlaying ensemble average currents were produced by convolving the derivative of the FL with or without a prepulse (C), respectively, and the Poo obtained with a prepulse (Poo, +pre), as explained in Results. D, Poo distribution. Smooth curve through the data are reproduced from Figure 2D. E, Open time histograms were fit by the sum of two exponentials with the following parameters:
Strong G-protein modulation was immediately evident in ensemble average currents (Fig. 3B), as the waveform observed without a prepulse was initially small and exhibited a slow phase of activation ("kinetic slowing"; Luebke and Dunlap, 1994
Averages from multiple patches confirmed the features suggested above by exemplar single-channel sweeps. First, the existence of a large fraction of slow-activating sweeps is supported by the slow component of the FL without prepulse (Fig. 3C, gray curve). Such sweeps are virtually absent after a prepulse, as evident from the fast and uniform rise of the corresponding FL curve (black curve), which is closely similar to those in control (Fig. 2C). Second, the persistence of a sparse gating mode in sweeps without prepulse is evident from the maintained deficit of the corresponding conditional open probability (Poo) (Fig. 3D, gray trace), compared with the Poo computed for sweeps with prepulse (black trace). The virtual absence of sparse gating after prepulse is established by the close similarity of this Poo for sweeps with prepulse (Fig. 3D, black trace) and those for control sweeps (Fig. 3D, fitted curve). Third, the indistinguishability of the dense-burst gating in sweeps with and without prepulse during G-protein modulation, as well as in control patches, is corroborated by the near identity of open and closed time distributions for the various conditions (Figs. 2, 3, panels E and F). Although computed from all events, these distributions are largely identical to those that would be obtained from pure dense-burst gating, because the sparse gating pattern contributes a comparatively negligible number of open and closed events (Fig. 3A, left column). Many of these single-channel characteristics have been previously observed and shown to account for the characteristics of macroscopic current waveforms (Patil et al., 1996
The important new finding came with the sparse gating pattern observed above (Fig. 3A, left column, sweeps 2, 3, 4), which featured openings with properties expected of the reluctant events whose existence we sought to determine. First, sparse gating was only appreciable where G-protein inhibition was expected to favor channel occupancy of the reluctant mode: present in modulated patches when observed without a prepulse, and essentially absent after a prepulse, or in control patches (where little basal modulation is present; Colecraft et al., 2000
Beyond supporting the existence of reluctant gating, the single-channel data could also provide in-depth kinetic information about such gating in isolation, after visual selection for sweeps (without prepulse) that started out in the sparse gating pattern. Lifetime distributions for the first openings of selected sweeps, which should mainly reflect reluctant gating, indicate brief, clearly submillisecond open times (
Impact of reluctant openings of N-type channels at physiological potentials
Indeed, the remaining challenge for proposals of a physiological contribution of reluctant openings was that they might not activate fast enough to contribute during the brief 1-4 msec duration of neuronal action potentials; the slow phase of the FL distribution that tallies the first occurrence of such openings (Fig. 3C, gray) fits a time constant of 116 msec. However, the +45 mV voltage step used in Figure 3 corresponds to
Figure 4 summarizes the results of single-channel experiments on inhibited N-type channels, using voltage steps to +60 mV (corresponding to +10 mV in 2 mM Ca2+). The protocols and format are otherwise identical to those in Figure 3. Despite the decrease in unitary current amplitudes (
0.5 pA) caused by the stronger depolarization, single-channel activity was still clearly resolved, including the sparse gating pattern believed to correspond to reluctant gating (Fig. 4A, left column, sweeps 1, 2, 4, and 5). As observed previously with depolarizations to +45 mV (Fig. 3), all of the data and analysis here (Fig. 4) supported the identification of sparse gating as reluctant mode activity, as well as the same elementary basis for G-protein modulation. Two important quantitative differences concerned the increased likelihood of evoking reluctant gating at physiological potentials. First, the steady-state probability of reluctant opening was now increased to ~0.07 (Fig. 4H, reluctant), consolidating projections favoring an appreciable reluctant open probability near the peak of action potentials (Fig. 1C, triangles). Second, the activation of presumed reluctant openings was markedly accelerated, as evident from exemplar traces (Fig. 4A, left) and the near coalescence of slow and rapid phases of the FL distribution (Fig. 4C, gray). At even greater depolarizations approaching action potential peaks, the activation of reluctant openings would be accelerated still further. Whereas single-channel experiments could not be performed explicitly at these higher physiological potentials, because of reduced signal amplitude, extrapolation of the results in Figures 3 and 4 strongly established the likelihood that reluctant N-type channel openings would contribute during neuronal action potentials.
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Figure 4. Reluctant N-channel openings occur with increased frequency and faster activation kinetics at +60 mV. A-H, Identical format as in Figure 3. A, Mean unitary current amplitude was 0.46 ± 0.01 pA (n = 3). B, Ensemble average currents. Identical format as in Figure 3B. C, FL distributions obtained either without (gray trace) or with (black trace) a prepulse. D, Smooth curve through the data are a least-squares fit of a biexponential function to the +prepulse data. E, Fits to open time histograms had the following parameters:
Before considering subsequent experiments that tested directly for such a physiological contribution, we next turned to single-channel experiments that established fundamental differences in the G-protein modulation of P/Q-type channels. This order permitted subsequent, side-by-side assessment of the physiological impact of differences in the behavior of these two channel types.
Absence of reluctant openings in G-protein-inhibited P/Q-type channels
On the basis of previous whole-cell experiments (Colecraft et al., 2000
To directly investigate these potential differences, we conducted experiments on single P/Q-type channels during G-protein inhibition (Fig. 5), using the identical protocols and format as with N-type channels (Fig. 3). The ensemble average current of P/Q-type channels displayed clear-cut kinetic slowing and prepulse facilitation (Fig. 5B), indicating strong G-protein modulation. Nevertheless, close inspection of ensemble currents indicated that the degree of inhibition of P/Q-type channels was clearly less than that obtained for N-type channels (Fig. 3B), demonstrating that the weaker inhibition of macroscopic currents has its basis at the level of individual channels. Single-channel records (Fig. 5A) contrasted sharply from those of N-type channels, in that there was no hint of the sparse gating pattern attributed to reluctant openings (Fig. 5D), despite clear delays in the onset of first opening in sweeps without prepulse (Fig. 5A, traces 1, 2, 4, and 5; also Fig. 5C). Gating subsequent to first opening always followed a dense-burst pattern that was unchanged by prepulse and identical to that in unmodulated controls, as established explicitly by the overlay of Poo for the various conditions (Fig. 5D) and the similarity of duration histograms (Fig. 5E,F).
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Figure 5. G-protein-inhibited P/Q channels display a delayed latency to first opening, with no evidence of reluctant openings. A-F, Identical format as in Figure 3. A, Mean unitary current amplitude was 0.64 ± 0.04 pA (n = 3). D, Smooth curve through the data are a biexponential fit to Poo generated from
Convolution analysis (Aldrich et al., 1983
Because of the faster gating kinetics of P/Q-type channels, it was not possible to routinely obtain well resolved, single-channel records at +60 mV, as was possible for N-type channel (Fig. 4). However, the following macroscopic current experiments confirmed the relative paucity of reluctant openings of P/Q-type channels at voltages greater than +45 mV.
Physiological impact of the differential occurrence of reluctant openings
To explore the physiological implications of the contrasting prevalence of reluctant openings in N-type and P/Q-type channels, we investigated the effects of G-protein inhibition of whole-cell Ca2+ currents evoked by action potential waveforms. If reluctant openings of N-type channels activate fast enough to occur during neuronal action potentials, their briefer open times and lower open probability suggests that G-protein inhibition would serve to both decrease the amplitude and alter the shape of Ca2+ entry waveforms during physiological stimulation. By contrast, the absence of reluctant openings in P/Q-type channels predicts that G-protein modulation would simply reduce the number of functional channels, resulting in decreased amplitude of the Ca2+ entry waveform without shape alteration.
Direct tests of these predictions are summarized in Figure 6. Whole-cell Ca2+ currents were elicited by action potential waveforms modeled after those recorded in the calyx of Held (Borst et al., 1995
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Figure 6. Functional impact of reluctant openings on Ca2+ currents evoked by neuronal APWs. A, APWs of different half-width amplitudes (1.5, 2, and 2.5 msec) used to elicit Ca2+ currents. B, Exemplar N-type currents elicited by the indicated APWs either under control conditions ( t90) in the presence or absence of CCh was measured (arrows).
To test for effects on the shape of Ca2+ entry waveforms, we scaled up +CCh traces (gray traces) to match peak amplitudes with those of
DISCUSSION
This study provides the first single-channel comparison of N- and P/Q-type calcium channels during G-protein inhibition. Single, inhibited N-type channels exhibited a sparse pattern of opening, which likely represents direct resolution of gating in the reluctant mode with G
Previous studies of reluctant gating
Several whole-cell studies have provided indirect evidence that N-type channels can open in the reluctant mode during G-protein inhibition (Elmslie et al., 1990
Moreover, our current study provides the first report of single P/Q-type channel gating during G-protein inhibition. Our results directly establish the absence of reluctant gating in P/Q-type channels, irrespective of voltages spanning the entire physiological range. An important caveat here is that we might not be able to detect reluctant P/Q-type channel openings should they occur much too slowly to impact currents evoked by action potential waveforms. Such a scenario, however, would be inconsistent with our previous whole-cell data (Colecraft et al., 2000
Features of channel structure underlying differing prevalence of reluctant gating
Much research has sought to determine the structural determinants of G-protein inhibition of neuronal calcium channels, using channel mutations and chimeras to identify critical regions on the corresponding pore-forming
Whether these regions all contribute similarly to G-protein inhibition across neuronal channel types seems less certain, given our finding that N-type, but not P/Q-type channels can open in the reluctant mode. Within the context of the willing-reluctant model of channel inhibition (Elmslie et al., 1990
Physiological implications of reluctant openings
The differential occurrence of reluctant openings in N-type versus P/Q-type channels raises rich possibilities for the dimensions of synaptic modulation produced by G-protein inhibition, especially because the involvement of these two channel types differs at spatially distinct synapses (Luebke et al., 1993
Our results suggest that at synapses where P/Q-type channels trigger release, presynaptic channel inhibition by G-proteins is attributable solely to delayed activation kinetics of inhibited channels (Fig. 5), effectively silencing them for the brief duration of neuronal action potentials. Hence, G-protein inhibition would reduce the amplitude of the waveform of Ca2+ entry during action potentials but preserve waveform shape (Fig. 6C). In this case, synaptic efficacy would be reduced, but the timing and precision of neurotransmission (Sabatini and Regehr, 1999
At synapses dominated by N-type channels, the effects of G-protein inhibition on synaptic communication could be considerably different. Here, the slower activation of inhibited channels would also cause many of them to remain closed throughout a given action potential, but a significant fraction of inhibited channels would contribute reluctant openings during a neuronal spike. Accordingly, whereas the silencing of inhibited channels would diminish the overall amplitude of Ca2+ entry, the faster deactivation kinetics pertaining to reluctant openings (Colecraft et al., 2000
Another potential effect of reluctant openings is to modulate the coupling efficiency of open N-type channels to release sites. The effect can be understood by considering that individual channels support a "calcium microdomain" surrounding the cytoplasmic mouth of the channel pore. At many synapses, the overlap of several domains with a single release site enables multiple channels to trigger the cognate release site (Mintz et al., 1995
Overall, these distinctions in G-protein inhibition of N- and P/Q-type channels contribute to the growing awareness that differences in the operational features of seemingly similar neuronal calcium channels, and their splice variants, may provide an important basis for custom regulation of synaptic function.
FOOTNOTES Received Oct. 11, 2000; revised Dec. 4, 2000; accepted Dec. 11, 2000.
This work was supported by grants from the National Institutes of Health (D.T.Y.) and a National Institutes of Health Medical Scientist Training Program Award (D.L.B.). We thank SIBIA Neurosciences for the human
Correspondence should be addressed to David T. Yue, Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205. E-mail: dyue{at}bme.jhu.edu.
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