Voltage-dependent facilitation of a neuronal - Europe PMC

The EMBO Journal vol.13 no.21 pp.5032-5039, 1994

Voltage-dependent facilitation of acC L-type calcium channel

a

Emmanuel Bourinet, Pierre Charnet', W.Jeffrey Tomlinson, Anthony Stea, Terry RSnutch2 and Joel Nargeot1

Hess, 1990; Richard et al., 1993), voltage-dependent phosphorylation (Artalejo et al., 1992; Sculptoreanu et al., 1993a,b) and a voltage-dependent deinhibition of tonic Gprotein block (Bean, 1989; Elmslie et al., 1990; Kasai, 1991). In the nervous system, the dissection of facilitation has been made difficult by the existence of multiple subtypes of Ca channels in many central neurons and by the inaccessibility of some Ca channels. Electrophysiological and pharmacological analyses have distinguished five major types of Ca channel expressed in neurons (T, L, N, P and Q types; reviewed by McCleskey and Schroeder, 1991; Zhang et al., 1993). T-type channels require hyperpolarization to remove inactivation and transiently activate with small membrane depolarization (low voltage-activated). The other four types are activated by stronger depolarization (high voltage-activated) and display diverse kinetics and pharmacological characteristics. L-type channels are sensitive to dihydropyridine agonists and antagonists, while N and P types are blocked by low concentrations of the peptide toxins, wo-conotoxin GVIA and w-agatoxin IVA, respectively. Q-type channels have been described only recently and may account for a large fraction of presynaptic neurotransmitter release (Wheeler et al., 1994). Most central neurons coexpress multiple Ca channel subtypes and immunohistochemical evidence indicates that they are differentially distributed at the subcellular level (Westenbroek et al., 1992, 1993; Hell et al., 1993). In recent years biochemical and molecular genetic analyses have begun to provide an insight into the primary structure and subunit composition of voltage-gated Ca channels. The skeletal muscle L-type Ca channel is a large hetero-oligomeric complex consisting of five subunits ((Is, °(2' 1la' y and 6; Campbell et al., 1988; Catterall et al., 1988). Exogenous expression studies using cDNAs show that cls forms a voltage-sensitive, Ca-selective pore and is the target of L-type Ca channel antagonists (PerezReyes et al., 1989). The other subunits appear to play roles in mediating the xls subunit physiological properties, with the la subunit having the most significant effects in coexpression studies (Lacerda et al., 1991; Varadi et al., 1991). To date, five cxl subunit genes (lIA, OCIB, XCC aID and OCIE) and four E subunit genes (PI. P2, 13 and N4) have been shown to be expressed in the nervous system (Castellano et al., 1993a,b; Soong et al., 1993; reviews by Tsien et al., 1991; Snutch and Reiner, 1992). Physiological and immunohistochemical studies demonstrate that aCB is the major subunit of the N-type channel complex, while the alC and OCID subunits are components of distinct neuronal L-type channels (Dubel et al., 1992; Westenbroek et al., 1992; Williams et al., 1992a,b; Fujita et al., 1993; Hell et al., 1993; Stea et al., 1993; Tomlinson et al., 1993). The OC1A subunit shares some functional properties with both Q- and P-type channels, while aXIE encodes a

Biotechnology Laboratory, Room 237, 6174 University Boulevard, University of British Columbia, Vancouver, BC, Canada V6T IZ and 'CRBM-CNRS UPR 9008, INSERM U249, 1919 Route de Mende, BP 5051, F-34033 Montpellier Cedex, France 2Corresponding author Communicated by P.de Camilli

Calcium entry into excitable cells through voltagegated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal axc L-type calcium channel can be facilitated by positive pre-depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage-dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G-protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel 1 subunit. In contrast, three neuronal non-L-type calcium channels, OClA, OCIB and alE, were not subject to voltage-dependent facilitation when coexpressed with a 1 subunit. The results indicate that the mechanism of neuronal L-type calcium channel facilitation involves the interaction of ax and A subunits and is dependent on protein kinase A activity. The selective voltagedependent modulation of L-type calcium channels is likely to play an important role in neuronal physiology and plasticity. Key words: calcium channel/dihydropyridine/facilitation/ protein kinase AlXenopus oocyte

Introduction Voltage-gated calcium (Ca) channels are found in most excitable cells where they contribute to both electrical properties and Ca-dependent processes such as neurotransmitter release and gene expression (reviewed by Tsien et al., 1988). An intriguing aspect of Ca channels is their plasticity and modulation. The voltage- and frequencydependent up-regulation (facilitation) of Ca currents (Fenwick et al., 1982; Hoshi et al., 1984; Elmslie et al., 1990; Pietrobon and Hess, 1990; Artalejo et al., 1992; Richard et al., 1993) has been implicated in a number of physiological processes, including increased muscle contractile force (Sculptoreanu et al., 1993a) and the secretion of catecholamines associated with the fight or flight response (Artalejo et al., 1994). Various mechanisms have been proposed for facilitation, including a switch between gating modes of channel activity (Pietrobon and

neuronal

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L-type Ca channel facilitation

novel type of neuronal Ca channel (Soong et al., 1993; Zhang et al., 1993; Stea et al., 1994). The expression of cloned neuronal Ca channel subunits in reconstitution systems such as Xenopus oocytes allows the functional characterization of individual Ca channel subtypes in isolation from other neuronal conductances. In this study we took advantage of this powerful method to show that of four cloned neuronal Ca channels tested, only the xlc L-type Ca channel is facilitated by the application of positive predepolarizations (PPs). Facilitation required coexpression of xlc and ,B subunits and was dependent upon protein kinase A (PKA) activity.

Results Functional neuronal L-type Ca channels were reconstituted in Xenopus oocytes by nuclear injection of expression plasmids containing cDNAs for rat brain xi,c, x2 and 1lb Ca channel subunits. During a typical test pulse from -80 to 0 mV in 40 mM Ba recording solution the cxlc + a2+ 1lb Ba current ('Ba) displayed very little inactivation (Figure la, trace 0). The application of positive prepulses (PP) of various durations (50-600 ms; Figure Ia) or intensities (+60 to + 140 mV, Figure la and b) induced a marked increase in the IBa amplitude during a subsequent test pulse. The kinetics and maximum amplitude of the facilitation were strongly voltage- and time-dependent (Figure Ib). For 800 ms PPs from +60 to + 120 mV the facilitated current increased between 170 and 390%. The time to maximal facilitation decreased when the PP level increased from +60 to + 120 mV (T = 300 to 78 ms, respectively; Figure 1 b). While under normal conditions, IBa exhibited very little inactivation (< 10%, T 4000 ms), the facilitated IBa (600 ms PP to + 140 mV) showed a fast and a slow inactivating component (T0ff = 298 and 4000 ms, Figure Ic), similar to that found for facilitated native cardiac L-type channels (Pietrobon and Hess, 1990). The rate constants of the onset of facilitation (I/TOn) and the inactivation of the facilitated current (l1/off) were also strongly dependent on the membrane voltage (Figure ld). Single channel recordings from cell-attached patches in the presence of Bay K8644 showed that the major effect of facilitation was an apparent increase in the probability of opening, as displayed by a decrease in the number of blank sweeps during the test pulse and the apparent increase in the number of open channels after the PP (Figure le). Facilitation did not alter the unitary current of the xlc L-type current (0.77 ± 0.09 and 0.79 ± 0.08 pA with and without PP, respectively). The magnitude of the amplitude increase seen with the ensemble average of single channels with and without PP (Figure le, bottom) is similar to the facilitation observed for macroscopic IBa in the presence of Bay K8644 (Figure 2b). Overall, the results show that neuronal Qxlc L-type currents expressed in Xenopus oocytes can be facilitated by PPs similar to the voltage-dependent facilitation recorded in cardiac cells (Pietrobon and Hess, 1990). In all of the experiments described, intra-oocyte Ca levels were efficiently buffered by BAPTA injection (see Materials and methods), suggesting that Ca-dependent enzymes, such as protein kinase C or calmodulindependent kinase II, were unlikely to underlie facilitation. The injection of GTPyS or GDP,S into oocytes during -

the course of the experiments did not alter either the amplitude of the control current without prepulse (not shown) or the extent and kinetics of the voltage-dependent facilitation (Figure 2a), suggesting that a direct G-protein interaction was not involved in clc facilitation. Inhibition of dephosphorylation using either the ATPyS or phosphatase inhibitor okadaic acid (OA) was also without significant effect (Figure 2a), and unlike the situation in chromaffin cells (Artalejo et al., 1992) did not maintain facilitation when prepulses were eliminated (data not shown). In contrast, incubation (12-16 h) in the presence of a non-selective kinase inhibitor (H7, 100 gM) resulted in a significant (P < 0.05) decrease in the magnitude of facilitation (-61 + 25%). Similarly, the injection of either of the protein kinase A inhibitors, A-PKi (Fernandez et al., 1991) or Rp-cAMP, significantly reduced both the amplitude of the basal current (by 37 ± 21%, n = 7 and 41 + 23%, n = 9, respectively) and the magnitude of the facilitation (by 73 ± 19 and 81 ± 16%, respectively; Figure 2a). Facilitation was not altered significantly by the injection of cAMP alone (data not shown), or in combination with the catalytic subunit of PKA and ATP (Figure 2a). Taken together, these results suggest that phosphorylation by PKA of the channel complex or associated proteins is necessary for the development of neuronal xlc L-type Ca channel facilitation and that the xlc channel is basally phosphorylated in oocytes. The control and facilitated currents were blocked completely by the Ca channel antagonists Cd (10 ,uM) and PN 200-1 10(10 (IM; Figure 2b), eliminating the possibility of involvement of the endogenous oocyte, the dihydropyridine-resistant Ca channel or other contaminating inward currents. Facilitation was still observed in the presence of the L-type agonist Bay K8644, although to a lesser extent (-24% increase, n = 4; Figure 2b). Microinjection of the divalent ion-chelating agent, BAPTA, completely suppressed the oocyte endogenous Ca-activated Cl current and permitted Ca to be used as the permeant ion through Ixlc L-type channels. Similar to that observed for L-type currents in native cells, the acx Ca current displays a fast Ca-dependent inactivation in the absence of PPs (Figure 2c). Using Ca (Figure 2c, n = 8) or Sr (n = 3; data not shown) as the charge carriers, the application of PPs was still able to elicit facilitation, indicating that unlike the frequency-dependent facilitation of L-type Ca current in cardiac cells (Fedida et al., 1988; Richard et al., 1993; Tiaho et al., 1994), voltage-dependent facilitation of the neuronal alc L-type Ca channel was independent of the permeating ion and the kinetics of inactivation. Indeed, attempts to induce frequency-dependent facilitation by increasing the rate of stimulation (stepping from -80 to +10 mV from 0.066 to 1 Hz) had no effect on the clc Ca currents (data not shown). A major factor regulating the development of facilitation is the subunit composition of the expressed channel (Figure 3). L-type current facilitation could be recorded in oocytes injected with combinations of axc + x2 + 1lb and cxc + lb, but not in oocytes where 1lb was absent. In addition to increasing the magnitude of whole-cell currents, PPs also caused a hyperpolarizing shift of the activation curve (AV0 5) and an increase in the voltage dependence of activation (increase in steepness expressed as A slope;

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Fig. 1. Activity of the neuronal acxr L-type Ca channel is potentiated by membrane predepolarization. (a) Ca channel activity was recorded in 40 mM Ba solution during a 200 ms test pulse to 0 mV from a holding potential of -80 mV (trace 0). A predepolarization to + 140 mV of various durations (50, 100, 200, 300, 400 and 600 ms) induced a marked increase (facilitation) in the amplitude of the subsequent Ba current recorded during normal test pulse. The facilitation reached a steady state for PP durations :600 ms when elicited at + 140 mV (calibration bars: 700 nA and 90 ms). (b) The steady state amplitude as well as the kinetics of the onset of the facilitation are strongly voltage-dependent. At each PP voltage the onset of facilitation (expressed as the current amplitude increase after PP of various duration over the current without PP) followed a sigmoidal time course. (c) In normal conditions (without prepulse) IBa does not display any significant inactivation, even during long test pulses (