Beta-adrenergic stimulation selectively inhibits long-lasting L-type calcium channel facilitation in hippocampal pyramidal neurons

Abstract

L-type calcium channels are abundant in hippocampal pyramidal neurons and are highly clustered at the base of the major dendrites. However, little is known of their function in these neurons. Single-channel recording using a low concentration of permeant ion reveals a long-lasting facilitation of L-type channel activity that is induced by a depolarizing prepulse or a train of action potential waveforms. This facilitation exhibits a slow rise, peaking 0.5-1 sec after the train and decaying over several seconds. We have termed this behavior "delayed facilitation," because of the slow onset. Delayed facilitation results from an increase in opening frequency and the recruitment of longer duration openings. This behavior is observed at all membrane potentials between -20 and -60 mV, with the induction and magnitude of facilitation being insensitive to voltage. beta-Adrenergic receptor activation blocks induction of delayed facilitation but does not significantly affect normal L-type channel activity. Delayed facilitation of L-type calcium channels provides a prolonged source of calcium entry at negative membrane potentials. This behavior may underlie calcium-dependent events that are inhibited by beta-adrenergic receptor activation, such as the slow afterhyperpolarization in hippocampal neurons.

Fig. 2.

A train of action potential waveforms induce delayed facilitation of L-type channels. Selected traces of 5 sec sweeps of channel activity for control (left) and after a train of action potential waveforms (right) at −20 mV. The patch appeared to contain two channels. Low P(o) activity was seen in control sweeps. Delayed facilitation was observed after the 50 Hz. train of an action potential waveform (inset). Each action potential waveform consisted of a ramp to a peak of +50 mV, followed by successive ramps to +20 and −10 mV. The potential was returned to −70 mV by an additional ramp (to mimic an afterhyperpolarization). The membrane potential was slowly ramped back to −60 mV before the next waveform was initiated (see Materials and Methods for details). Delayed facilitation was evoked by 10 action potential waveforms in 200 msec, giving a frequency of 50 Hz. Openings with a curtailed amplitude reflect short duration openings with apparent amplitude that is clipped at the bandwidth used (2 kHz). Expanded traces for behavior observed in control jumps and after the train of action potential waveforms are shown below. The expanded traces were taken from the sweeps marked with a bar and *. As is seen in open duration histograms (see Fig. 6), delayed facilitation is caused by an increase in opening frequency and an increase of longer duration openings.

Fig. 1.

Delayed facilitation of L-type calcium channel activity by a depolarizing prepulse. A, Stability plot of L-type channel activity in a cell-attached patch, showingP(o) for each 6 sec sweep. The patch appeared to contain a single channel. The patch potential was stepped to −30 mV for 6 sec from a holding potential of −60 mV, either directly (Control, thin bar) or immediately after a prepulse to +40 mV (200 msec duration) (Prepulse, thick bar). Channel activity was low throughout the control sweeps and was dramatically augmented after the depolarizing prepulse. Sweeps were considered facilitated if the patch P(o) measured over the entire 6 sec sweep exceeded two times the mean P(o) of control sweeps (dashed line). B, Selected sweeps as indicated inA. Little channel activity was observed on changing the patch potential from −60 to −30 mV (control, left traces). After the prepulse to +40 mV, there was a large increase in L-type activity. Short duration events were observed throughout the 6 sec sweep.

Fig. 3.

Trains of action potential waveforms are more suited to promoting delayed facilitation than a depolarizing prepulse.Top, Time plot of patch P(o) for each 6 sec postpulse potential of −50 mV. Holding potential was −60 mV. The patch appeared to contain two channels. Delayed facilitation was not observed with a 200 msec prepulse to +40 mV but was observed with a 50 Hz train of an action potential waveform (see Fig. 2,inset, for waveform schematic and Materials and Methods for description). Bottom, Selected traces of the 6 sec time segment used to calculate patch P(o). LowP(o) activity was seen for both control and after a prepulse. Delayed facilitation was observed after the 50 Hz train of an action potential waveform.

Fig. 6.

Time-dependent increase in P(o) results partly from an increase in long duration openings.A, Top, Open probability in 200 msec time segments averaged over a 5 sec voltage pulse to −20 mV. Under control conditions (mean of 10 sweeps) the P(o) was low throughout the voltage pulse (left). A 200 msec prepulse to +40 mV (right) caused a dramatic increase inP(o) with a rise time of ∼600 msec and a subsequent decay (mean of nine facilitated sweeps). The rising phase of the waveform was interpolated with a continuous line, and the decay was fit with an exponential function (τ ∼1.5 sec).Bottom, Open duration histograms show that openings under control conditions were of very short duration (τ ∼0.4 msec) (0% of long openings), and that the prepulse recruited longer duration openings (13% of events), best fit by an exponential of τ ∼4 msec.B, Top, P(o) plots of control (mean of 14 sweeps) and after facilitation (mean of 10 sweeps) measured at −50 mV. As seen at −20 mV, a prepulse induced a time-dependent increase inP(o). The rising phase of the waveform was interpolated with a continuous line, and the decay of facilitatedP(o) increase was fit with an exponential function (τ ∼1.3 sec). Bottom, Open duration histograms show that the prepulse-induced increase in P(o) was accompanied by an increase in longer duration openings (long duration openings contributed 6% of events in control sweeps, which increased to 11% in facilitated sweeps). Holding potential was −60 mV for both patches.P(o) measurement began 250 msec after the prepulse to exclude the fast decaying form of prepulse facilitation (see text).

Fig. 8.

Inhibition of delayed facilitation by β-adrenergic receptor stimulation. Cell-attached patch recording of delayed facilitation of L-type channel activity. The patch appeared to contain two channels. A, Top, Under control conditions, a 200 msec prepulse to +40 mV induced facilitation of L-type channel openings at −40 mV. Bottom, Open duration histogram of all events after the prepulse. The distribution was best fit by the sum of two exponentials with time constants 0.3 and 2.5 msec.B, Top, In the presence of the β-adrenergic agonist isoproterenol (1 μm), the prepulse failed to evoke high P(o) L-type channel activity.Bottom, Open state analysis of events after the prepulse were best fit by a single exponential distribution (τ ∼0.3 msec).C, Top, The β-adrenergic receptor antagonist propranolol (10 μm) was applied in the continued presence of isoproterenol. Approximately 5 min after the antagonist was added, delayed facilitation was observed after the prepulse.Bottom, Open duration histogram of events after the prepulse, in the presence of isoproterenol and propranolol. Recovery from the effect of isoproterenol was apparent with the return of the longer time constant (τ ∼3.1 msec).

Fig. 5.

Ensemble current of delayed facilitation induced by a train of voltage pulses. Ensemble current generated by a 100 Hz train of rectangular voltage pulses to +40 mV of 5 msec duration, separated by 5 msec intervals. Inset, Voltage protocol in more detail. Holding potential was −60 mV, and post-train voltage was −30 mV. Ensemble is an average of 27 sweeps. A slow rise of inward current was observed, peaking ∼600 msec after the termination of the train. The rising phase of the waveform was interpolated with acontinuous line, and the decay was fit with an exponential time course (the arrow marks the start point of the exponential fit; τ ∼1.6 sec). The dashed linerepresents zero current.

Fig. 4.

Channels underlying delayed facilitation are sensitive to the DHP agonist BAY K 8644. A, Inward channel openings evoked by repeated depolarizing voltage pulses from −60 mV to 0 mV recorded from a cell-attached patch with 10 mm Ba²⁺ as the charge carrier. Channel openings are downward. This patch appeared to contain two channels. i, In the absence of a DHP agonist, single-level channel openings were brief and were observed throughout the 200 msec depolarization. Generation of an ensemble current (average of 23 sweeps) gave a waveform that showed little decay during the depolarization (iii). ii, In the presence of the DHP agonist BAY K 8644 (5 μm), channel openings were of long duration and were observed to the second level. Generation of an ensemble current (average of 27 sweeps) showed that BAY K 8644 had greatly augmented channel activity (iv).B, Current–voltage relationship of channel amplitude observed in the absence and presence of BAY K 8644. Channel amplitude was obtained by gaussian fits to amplitude histograms obtained by visual inspection of each opening (see Materials and Methods) evoked by a family of depolarizing voltage pulses to −40 to 10 mV (holding potential, −60 mV). Continuous lines represent the least squares fit to the data. In the absence of BAY K 8644 (•), channel slope conductance was 10.5 pS. In the presence of BAY K 8644 (♦), the channel slope conductance increased to 15.5 pS (see Results). Channel amplitude observed in the absence of BAY K 8644 during 5 sec pulses to −20 (see below), with (▴) or without (▾) a train of action potential waveforms superimposed on the controlI/V. In the presence of BAY K 8644, the increase in channel amplitude was also observed for openings evoked during 5 sec pulses to −20 (see below), with (▵) or without (▿) a train of action potential waveforms. C, i, iii, Records evoked by a 5 sec voltage pulse from −60 mV to −20 mV (see Fig. 2 for protocol) in the absence (i) and presence (iii) of BAY K 8644 (5 μm). ii, iv, Records evoked by a 5 sec voltage pulse to −20 mV (holding potential, −60 mV) preceded by the train of action potential waveforms (see Fig. 2 for protocol) in the absence (ii) and presence (iv) of BAY K 8644 (5 μm).i, ii, Delayed facilitation was evoked by a train of action potential waveforms and was observed at −20 mV, with few openings seen in the absence of the action potential waveform train. Induction of delayed facilitation demonstrated that the patch contained two channels. iii, iv, In the presence of BAY K 8644, long duration openings were observed during 5 sec pulses to −20 (see below), with or without a train of action potential waveforms. Note the addition of BAY K 8644 caused the short duration openings to be replaced by openings characteristic of DHP-agonist modified channels. In addition, note that normal channel gating was observed between bursts of DHP-modified behavior (iv, upper trace).

Fig. 7.

Delayed facilitation of L-type channel activity is not markedly voltage-dependent. A, The contribution of the exponent with the longer time constant is plotted for control and facilitated sweeps. Longer duration openings contributed ∼1% of events in control sweeps. After facilitation by a 200 msec prepulse to +40 mV, longer openings constituted ∼5% of all events. The increase in the number of longer duration openings was significant at each voltage (one-way ANOVA, p < 0.01). This increase in longer duration openings was the same at each membrane voltage (n = 3–5 for each voltage). B, The magnitude of the increase in peak P(o), induced by a prepulse to +40 mV (200 msec duration), or a train of action potential waveforms was not obviously voltage-sensitive. The ratio of the peakP(o) observed in 200 msec time segments to the meanP(o) observed during the 6 sec control sweep is plotted as mean ± SEM. The effect of postprepulse displayed no obvious voltage dependence (one-way ANOVA, p > 0.1).C, The induction of delayed facilitation was not dependent on the postprepulse potential. The ratio of facilitated sweeps relative to the total number of sweeps is plotted. There was no significant difference in the number of facilitated sweeps observed at each potential (one-way ANOVA, p > 0.1).D, Open-state kinetics of L-type channels are not voltage-dependent. Plotted are time constants (short and long) obtained from the fitting of open duration histograms (for example, see Fig. 6). Time constants were obtained from control sweeps (open triangles, open circles superimposed with closed circles), facilitated sweeps (closed triangles, closed circles), and standard activation voltage pulses from a holding potential of −60 mV (closed and open diamonds). Channel open times obtained under all conditions were voltage-insensitive (n = 3–6 for each voltage).

Fig. 9.

Selective inhibition of delayed facilitation by β-adrenergic receptor activation or addition of a cAMP analog.A, The P(o) measured in 6 sec sweeps at −40 or −50 mV after a prepulse to +40 mV is normalized to control (without the prepulse). In the absence of isoproterenol, the prepulse induced a 5.0 ± 1.3-fold increase in P(o) during facilitated sweeps (n = 5). In different paired experiments (before addition of 8-CPT cAMP), the prepulse induced a 7.2 ± 0.83-fold increase in P(o) during facilitated sweeps (n = 4). The increase in patchP(o) was inhibited by the addition of the β-adrenergic receptor agonist isoproterenol (1 μm) or the membrane-permeant analog of cAMP 8-CPT cAMP (1 mm). Results are from paired data, with the effect of either isoproterenol or 8-CPT cAMP being compared with delayed facilitation evoked before their application. The two control bars, in isoproterenol and in 8-CPT cAMP, have been normalized to the first control (in the absence of treatment), showing that neither isoproterenol nor 8-CPT cAMP had an effect on control behavior. B, The P(o) observed during a 200 msec voltage pulse from −60 mV to 0 mV in the presence of isoproterenol was normalized to the P(o) observed in the absence of agonist. Isoproterenol had no significant effect on the channel activity observed during this standard activation protocol (n = 4).