Pathways of Ca2+ influx at the plasma membrane: Voltage-, receptor-, and second messenger-operated channels

Experimental

Cell Research 171 (1987) 271-283

REVIEW ARTICLE Pathways of Ca *+ Influx at the Plasma Membrane: Voltage-, Receptor-, and Second Messenger-Operated Channels JACOPO

MELDOLESI**’

and TULLIO

POZZAN?

*Department of Pharmacology, CNR Center of Cytopharmacology and Scientific Institute S. Raffaele, University of Milano, Milano, Italy, and bnstitute of General Pathology, CNR Center of Mitochondrial Physiology, Urkbersity of Padova, Padova, Italy

Accurate control of the cytosolic Ca2’ concentration, [Ca2+]i, represents a fundamental property of all living cells. Under resting conditions [Ca2+]i is set to values ranging (in various cell types) between 80 and 180 nM, i.e., about four orders of magnitude lower than the extracellular Ca2’ concentration, [Ca2’],. Stimuli of various nature cause [Ca2+]i to rise (often to values in the micromolar range), and the rise is in turn responsible for a wide spectrum of effects: activation of Ca2+-dependent enzymes located both in the cytosol and at the cytosolic surface of membranes; changes in membrane permeability; stimulation of integrated processes such as contraction, secretion (by exocytosis) of neurotransmitters, hormones and exocrine products; cell locomotion. Persistent, large elevations of [Ca2+]i have deleterious effects (contracture of the cytoskeleton; depolarization of mitochondria) and ultimately lead to cell death. Control mechanisms of [Ca2+]i are located both in the plasmalemma and within the cells [l-4]. Opening of plasmalemma pathways (channels) of various types causes Ca2’ to flow passively into cells, along its steep electrochemical gradient. Such an influx is balanced by active Ca2+ pumping (by a ubiquitous, [Ca2+]isensitive, calmodulin-containing plasmalemma enzyme) as well as by Ca2’ exchange with Na+ (via an electrogenic system present in most excitable cells, whose directionality appears governed by both [Na+]i and membrane potential [l-3]). Moreover, Ca*+ can be specifically segregated within membrane-bounded organelles: mitochondria [I] as well as a “microsomal,” high-affinity pumping store, whose cytological nature has not been identified with certainty yet. During the last few years, this second store has attracted a great deal of interest because of its ability to release Ca2’ (and thus to cause [Ca2’]i to rise, a process referred to as Ca*’ redistribution) in response to specific hormonal signals 14-61. The present article is focused on the various types of Ca” channels that are known to exist in eukaryotes. Specifically, we will deal with the channels that are gated by membrane potential (voltage-operated channels, VOCs) or activated as a consequence of neurotransmitter (or hormone) binding to specific receptors. ’ To whom reprint requests should be addressed at Department of Pharmacology, University of Milano, via Vanvitelli 32, 20129 Milano, Italy. 271

Copyright @ 1987 by Academic Press, Inc. All rights of reproduction in any form reserved 0014~4827/87 $03.00

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We propose to classify these latter channels into two groups: those where channel and receptor functions reside in the same molecule (receptor-operated channels, ROCs); and those (probably) activated by second messengers generated following receptor activation (second messenger-operated channels, SMOCs). The other regulatory processes of Ca*’ homeostasis, which have been mentioned above, will be considered only in passing (redistribution) or not at all (Ca” pump; Na+/Ca*+ exchange). Until a few years ago Ca *+ channels were mostly investigated by two’types of techniques: classical electrophysiology and 45Ca fluxes [7-91. Direct measurement of [Ca*+]i was limited to a few types of large cells that could be impaled or microinjected. The development of new experimental procedures, such as patch clamp electrophysiology [lo] and the techniques for measuring [Ca*+]i by means of fluorescent trappable Ca*’ indicators (quin 2 and fura 2 [ll, 12]), together with the refinement of biochemical studies and the application of recombinant DNA techniques, had a major impact in the field and has modified considerably our knowledge and understanding of the structure, functioning, and regulation of Ca*+ channels. This article is not conceived as a comprehensive description of this recent progress. Rather, its aim is to enlighten the state of a number of important problems: those that have been successfully tackled as well as many others that remain open or obscure. VOLTAGE-OPERATED

Ca*+ CHANNELS

For a number of years progress in the field of Ca*’ voltage-operated channels (VOCs), although considerable, was sketchy and problematical. On one hand, important results were obtained; for example, the existence of Ca*’ currents triggered by depolarization was unambiguously documented in a variety of excitable cell types (from oocytes to neurons) and specific, clinically important drugs (Ca2+ antagonists or Ca*’ channel blockers) were developed. On the other hand, conflicting results were obtained in different tissues (for example, in the heart with respect to the brain) and doubts remained as to some fundamental properties of the Ca*+ channel, for example, its mechanisms of activation and inactivation and its sensitivity to blocker drugs (for reviews see [7-9, 13-161). In retrospect, it appears that many of these problems were due to the heterogeneity of Ca*’ VOCs, which was only suspected at that time and has been proven since by patch clamp single channel recording. Duality of the Ca*+ VOCs might have been expected in view of the dual physiological role of Ca*+: a charge carrier and an intracellular signal. Indeed, channels that appear to play primarily electrical (pacemaking) or intracellular signaling ([Ca*‘]i rise) roles have been shown to coexist in various types of cells. In addition, Ca*’ VOCs of a third type have been recently discovered in neurons. Following the simple nomenclature proposed by Nowycky et al. [17] we refer to these three types of VOCs as the T, L, and N channels, respectively. At the present time it would be inappropriate to consider each of these as a homogeneous type of channel. In contrast, it appears likely that heterogeneity (probably to a considerable extent) exists within each type. This conclusion is supported also by very recent results with the o toxin of the snail

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Conus geographus, the only toxin among those investigated so far [18] that appears certainly addressed to Ca2’ VOCs. 51 toxin was found to irreversibly block both L and N (but not T) VOCs in neurons and to be inactive on Ca2+ VOCs of any type in nonneuronal preparations [ 191. T (transient) Ca2+ VOCs were initially described in chick dorsal root ganglion (DRG) neurons [201 and later detected in mouse DRG neurons, heart, skeletal and smooth muscle, and pituitary GH3 cells [21-251. In contrast, no T channels are present in rat sympathetic neurons [19, 261. Activation of these channels occurs by small depolarization from very negative potentials (low threshold channels) and is followed by rapid, voltage-dependent inactivation [17, 19-251. Because of both their transient opening time and their conductance, which is small, these channels are expected to play a prominent role in the initiation of action potentials (pacemaking) rather than in [Ca2+]i homeostasis. T channels are insensitive to dihydropyridines (DHP), the very potent Ca2+ channel-blocking drugs [ 15, 17, 271. Recently it has been reported that catecholamines (noradrenaline and dopamine) markedly inhibit T (as well as L) channels in chick DRG neurons [25]. The intracellular mechanism underlying this effect has not been investigated yet. L (long-lasting) Ca 2+ VOCs are the classical, widespread late channels and the target of DHP [13-17, 27, 281. Because of their high conductance and prolonged opening time, these channels appear involved primarily in the regulation of [Ca2’]i. In many cell types, activation occurs at membrane potentials more positive than -30 mV and is maximal around + 10 mV. Inactivation is slow and in many cells is greatly influenced by [Ca’+]i [7, 15, 28, 291. Our recent studies in neurosecretory PC12 cells loaded with trappable Ca2+ indicators [30], however, have demonstrated that L channels also inactivate when [Ca2+li is maintained at the resting level. Thus, inactivation of this channel appears to be controlled by a dual mechanism: a time- and voltage-dependent mechanism, and a [Ca2+]i-dependent mechanism. Studies in various cell types (discussed in Ref. [30]) have shown that during prolonged (minutes to tens of minutes) depolarization a fraction of the L-type VOCs remains activatable, and this leads to a persistent elevation of [Ca2’]i to a new steady state that represents the equilibrium between the increased permeability of the plasmalemma and the stimulated Ca2+ extrusion activity of the cell. Under these conditions, application of Ca2’ channel blockers rapidly brings [Ca2’]i down to the resting level. Whether activation and inactivation of L channels require net movement of charges across the plasma membrane, as previously demonstrated for the Na+ VOC [31, 321, remains controversial [7, 151. Alternative models have been proposed [281. An interesting property of L channels is their regulability by hormones and neurotransmitters [8, 13-15, 331. In the heart, Ca” currents were found to be modulated by treatments that modify the cellular level of CAMP, and thus affect the state of phosphorylation of the substrates of CAMP-dependent protein kinases [13-151. Recently, the Ca2’ VOC itself has been shown to be a substrate of both CAMP- and Ca2+-dependent protein kinases. Whether the phosphorylation of the channel directly accounts for the CAMP-induced modulation has not been established yet [34, 353. Alternatively, phosphoprotein(s) associated with the cytosolic

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surface of the channel might be the direct target of the modulation [36]. At the single channel level, the effect of CAMP consists of the increase of the opening probability of L channels, with no change of their kinetics and unitary conductance. The CAMP-dependent modulation accounts for both the positive and the negative inotropic effects of fi adrenergic and muscarinic agents, respectively. These agents operate through their specific receptors @?, adrenergic and Mz muscarinic receptors), whose opposite effects on adenylate cyclase (activation and inhibition, respectively) are mediated through the corresponding coupling proteins, Gs and Gi, regulated by the binding of guanine nucleotides [37, 381. Agents effective, no matter at what level, in the chain of events initiated by receptor activation will ultimately affect the regulation of cardiac L-type Ca*’ vocs [13-151. Regulation by CAMP-dependent phosphorylation(s) is by no means a general property of L-type Ca*’ VOCs. In various cell types, induction of CAMP increase was found to be without effect on Ca*+ currents. In contrast, in two lines of rat neurosecretory cells [39] (PC12 and RINmSF, derived from a pheochromocytoma and insulinoma, respectively), as well as in chick DRG neurons [40], L channels were found to be negatively modulated by activators of a different type of kinase, protein kinase C [41]. Physiologically, this enzyme is activated by diacylglycerol (DAG), a second messenger generated by another receptor-triggered reaction, the hydrolysis of membrane poly(phosphoinositides) (PPI) [4, 5, and below]. In DRG neurons this mechanism of Ca*+ channel modulation has been suggested to be responsible for the inhibitory effects induced by the activation of receptors (a2 and GABA B), working again through a G-coupling protein whose nature has not been established yet [42]. Similarly, in AtT20 cells (a line derived from a mouse ACTH-secreting tumor) somatostatin has been found to inhibit Ca*+ VOCs through a G protein, but the involvement of protein kinase C has not been reported [43, 441. L-type Ca *+ VOCs have been purified from a particularly rich source, the transverse tubules of rabbit skeletal muscle, and found to consist of a major, high-molecular-weight (140K) integral membrane protein accompanied by two smaller subunits [45-47]. Whether this protein accounts for L channels in all cell types, however, is doubtful because recent reconstitution experiments [47, 481 demonstrated that the skeletal muscle channel has features (unitary conductance, kinetics) quite different from those of the heart channel [48]. In addition, two types of L channels (or, alternatively, two functional states of the same channel), both inhibitable by DHP drugs, have recently been reported to exist in skeletal muscle cells [24]. The possibility that L channels are a family, rather than a homogeneous type, of VOC appears therefore quite likely at the present time (Fig. 1). The third type of Ca*’ VOC, the N channel, is activated by strong depolarization from negative holding potentials. Its conductance and opening time appear intermediate between those of L and T channels. Like T channels, N channels are insensitive to DHP [17]. At present, considerable interest exists about this VOC because preliminary results suggest its involvement in the regulation of

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Fig. 1. Section through the hypothetical representation of a Ca” VOC. In the high-molecularweight integral membrane glycoprotein (Y stands for a sugar chain) the following regions are indicated: J=negatively charged selectivity filter; 2=gates with adjacent; 3=voltage sensors. Binding sites for Ca*’ and various phosphorylation sites are located at the negatively charged cytosolic (in) surface of the VOC to account for [Ca’+]i-dependent inactivation and various protein kinase-dependent modulations.

neurotransmitter release at nerve terminals and neurosecretory cells [ 151. So far, channels of the N type have been experimentally demonstrated in chick and rat DRG and in rat sympathetic neurons [15, 17, 26, 491. In rat DRG neurons L- and N-type channels appear to be concomitantly inhibited by GABA B-receptor activation [49]. In contrast, in SCG neurons only N channels are modulated by acetylcholine working through a muscarinic M1 receptor. Interestingly, this selective effect, although mediated by a G protein, can be duplicated by neither CAMP nor diacylglycerol[26]. Thus, a different mechanism (another second messenger; the direct interaction of N channel, G protein, and Ml receptor in the plane of the membrane) might be considered to account for these results. RECEPTOR-OPERATED

Ca2+ CHANNELS

Throughout the literature, the definition of Ca” receptor-operated channels (ROCs) is usually employed “at large,” i.e., to include all Ca” channels that are opened not by depolarization but as a consequence of the activation of various types of receptors. It occurs, however, that non-voltage-gated Ca2+ channels belong to two groups that indeed have very little in common. These groups are (1) ROCs sensu strictu, in which receptor and channel functions coexist in one single or two closely adjacent molecules and therefore where receptor activation and channel opening appear intimately interconnected, and (2) SMOCs, which are (or are believed to be) opened by second messenger(s) generated within the cells in response to receptor activation. These latter channels are discussed in the subsequent section. The nicotinic acetylcholine receptor will be considered as the ROC prototype. Clearly, this is not a pure Ca2+ channel, inasmuch as under physiological conditions it transports mainly Na+, as well as K+. However, because of its considerable Ca” permeability its discussion here appears appropriate, even if a detailed

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description of this fascinating pentametric glycoprotein, established by biochemical, morphological, and gene cloning studies, also carried out in combination with electrophysiological experiments [5&52], is beyond the scope of the present article. Among the most recent progress in this field that we would like to briefly mention are the cloning of the receptor, the demonstration of its heterogeneity in different cells (torpedo electric organ vs adult skeletal muscle receptors, with neuronal receptors also different from either one of these), and the initial characterization of its channel properties at the molecular level [52-551. In addition, it should be mentioned that the activity of this receptor (in particular its desensitization) appears under the control of phosphorylations 1561. The picture that emerges from these studies is astonishingly similar to that of the VOC for which detailed information is also available at the present time, the voltage-gated Na+ channel [31, 321. Thus the hypothesis might be proposed that VOCs and ROCs, although activated by different mechanisms, are not basically different entities but represent variations of a common, general theme. As far as the direct physiological role of the nicotinic acetylcholine receptor on Ca2+ transport, variable results have been obtained. In a subclone of PC12 cells (different from the subclone used in our laboratories) activation of this receptor has been reported to cause per se a rise of [Ca*‘]i sufftcient to evoke a secretory response [57]. Whether this occurs in other cell types, however, is doubtful. For example, in bovine chromaffin cells we recently found that the [Ca2+]i increase evoked by the activation of the nicotinic receptor disappears almost completely when Ca2’ VOCs are blocked by DHP drugs. The remaining [Ca2+]i rise, directly dependent on the activation of the nicotinic ROC, was minute and certainly subthreshold with respect to catecholamine release (unpublished results). The nicotinic channel is not the only ROC we know. Recent evidence has indicated that the brain glutamate receptor that has affinity for N-methyl-Daspartic acid (NMDA receptor) has a ROC structure and is permeable to both monovalent cations and Ca” [58]. This finding is important also because NMDA receptors are believed to play major roles in a number of physiological processes as well as in neurotoxicity. Other amino acid receptors of the brain (for example, glutamate receptors preferring quisqualic and kainic acids) might also be built according to a ROC scheme [58, 591. Thus, ROC sensu strictu appear as a stillgrowing family, and their physiological importance is expected to become better appreciated in the near future. Within the ROC group might also be grouped a channel described in mast cells, which is functionally related to the immunoglobulin Fc receptor. The antiasthmatic drug sodium dichromoglycate (DCG) was reported to bind specifically and to block this channel (DCG channel) and was therefore used as a tool for its isolation. The trigger for opening is believed to be the coclustering of DCG channels with antigen-activated Fc receptor [60]. Very recent electrophysiological studies, however, carried out by an elegant variation of the patch clamp whole cell mode, failed to substantiate the existence of a DCG channel [61, 621. In addition, it should be acknowledged that DCG channels, even if they exist, appear quite different from the other ROCs, as well as from the true SMOCs

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which, by the way, are not inhibited by DCG. The entire DCG channel story therefore appears open to question and might be better defined in the future.

SECOND

MESSENGER-OPERATED

Ca2+ CHANNELS

In a wide variety of cell types (possibly in all cells, no matter whether excitable or not), activation of a large number of receptors triggers, among other effects, a voltage-independent increase of the Ca2+ permeability of the plasma membrane. As discussed below, such an increased permeability might be due to the opening of channels triggered by second messengers generated by receptor activation. Thus, at these channels second messengers may play a role much more important than the modulatory role discussed above for Ca2’ VOCs. Based on this consideration we propose that these channels be lumped together under the (tentative) name of second messenger-operated Ca2+ channels (SMOCs). Although widespread, and possibly large, the group of Ca2” SMOCs is certainly the least exactly defined in the field of Ca2+ channels. Until very recently (September 1986), no electrophysiological studies had been reported and therefore the final proof of the channel vs carrier nature of the increased Ca2’ permeability remained to be given. The information available had been obtained almost exclusively by 45Ca transport experiments. Because of the small size of the influx responses, and because of the concomitant Ca2’ redistribution triggered by receptor activation (see below), these data were often of questionable value, especially when obtained after long (minutes to hours) incubations. In other, more appropriate experiments, increased 45Ca accumulation was demonstrated within a few seconds from receptor activation (see for examples Refs. [63-67]). The recent introduction of trappable fluorescent Ca” indicators [ 11, 121 has yielded new, but still indirect information on these channels. The increased Ca2+ permeabilities were found to be sustained (minutes to hours) and to lead to elevated [Ca2+]i steady states rapidly dissipated by specific receptor blockade [67-69]. Direct and probably definitive proof of the existence of bona fide SMOCs has now been reported in two cellular systems: T lymphocytes and neutrophils. In both these systems single-channel analysis carried out by the patch clamp technique has revealed small channels whose activation is totally independent of the membrane potential and occurs with some delay after application of the stimulation (presumably the time necessary for the generation of the second messenger) [70, 711. A question that might be asked is whether in a cell a single set of Ca” SMOCs is opened by the activation of various receptors. Alternatively, activation of each receptor type could trigger a separate set of SMOCs. Uncertainty in this field is due also to the fact that no drugs specifically addressed to SMOCs are known as yet. Although up to now only a few experiments have been carried out to tackle this problem, the results obtained appear to favor the first alternative [64-69, 72, 731, consistent also with the interpretation of SMOC activation that is discussed below. Receptors involved in Ca 2f SMOC activation are those coupled across the plasma membrane to the hydrolysis of PPI (Fig. 2). This is a large group of

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IA

Ca*+ pa*+

6

Ca”

L/

qvoc

~ATP~ 1 ,+DP+ Pi <-<

microsomal Nat

[Ca*+li

store\

~a*’

;Na*

ROC \A

I

at rest

: 1O-7 M

[Ca*+

10~10~3 M

Fig. 2. Schematic representation of a cell, with indication of the various processes involved in Ca2+ homeostasis. The Ca2+ pump and Na+/Ca2+ exchanger are indicated at the top, Ca2’ channels of the various types to the right. Also to the right is a receptor coupled to PPI turnover (R-PPI). Of the messengers generated by the activation of that receptor; inositol-1,4,5-trisphosphate (IP3) acts intracellularly to release Ca*’ loaded (by a pump mechanism) into a microsomal store adjacent to the plasmalemma. A second messenger (X) might cause the opening of Ca2+ SMOCs. Two alternatives (dotted arrows) are depicted for Ca2’ transport at that channel: direct loading into the microsomal store or release to the cytosol. Mitochondrial Ca2+ transport is also indicated.

receptors that comprises (in many, but not necessarily in all cells) the M1 muscarinic, al adrenergic, H, histaminergic, and many peptidergic receptors, such as the angiotensin II, bradykinin, neurotensin, vasopressin receptors and many others, including several mitogens [4, 5, 741. The very complex processes of transmembrane signaling and second messenger generation that are triggered at these receptors (Fig. 2) have been partially elucidated thanks to the pioneer work of Michell [74] and the following contributions of Berridge, Irvine, and other groups, in Europe and in the United States [4-6]. Receptor actiyation is known to cause the activation of an enzyme, a phosphodiesterase (phospholipase C), that preferentially cleaves a minor phospholipid of the plasmalemma inner leaflet, phosphatidylinositoL4,5bisphosphate (PIP2), with generation of two interesting metabolites, DAG and inositol-1,4,5trisphosphate (IP3). The receptor-enzyme interaction is mediated by a G-coupling protein (Gp, not yet characterized but apparently very similar to Gs and Gi [75]. Because of the different effects on PPI hydrolysis of a toxin, pertussis toxin, it appears likely that Gp is indeed a family of heterogeneous coupling proteins. The intracellular consequences of the stimulated hydrolysis of PPI are astonishingly large. Both of the two direct PIP2 metabolites appear to play second messenger roles. DAG is the specific activator of protein kinase C, a ubiquitous enzyme known to be

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involved in the regulation of a variety of important cellular functions [41], while IP3 is the mediator of Ca2’ redistribution from the high-affinity “microsomal” store to the cytosol[4, 51 (Fig. 2). Such a process is initiated by the binding of IP3 to a specific site at the cytosolic surface of the microsomal membrane [76], followed by the opening of a nearby channel, not yet characterized. By analogy with these processes it has appeared reasonable to assume also that the Ca2+ influx triggered by receptor activation is regulated by a second messenger generated, directly or indirectly, by PPI hydrolysis. So far, however, this second messenger has not been identified. Neither IP3 nor DAG appears to be implicated as the first causes Ca2+ release when applied to microsomes, but not to plasmalemma vesicles [77], while the second (as well as its analogs) causes no rise of [Ca2+]i when applied to intact cells (see from example [67,78]). Phosphatidic acid, which results from DAG phosphorylation, has been considered as a possible physiological Ca” ionophore [79], but a good demonstration has never been given, while theoretical considerations strongly argue against such a proposed role. The recent demonstrations that the PPI cycle is more complex than previously envisaged and includes successive phosphorylation and dephosphorylation of IP3, has introduced other possible candidates, inositol-I ,3,4,5tetrakisphosphate and inositol-1,3,6trisphosphate [80], which, however, have not been tested experimentally yet. In contrast, patch clamp evidence in favor of an activation by increased [Ca2+]i (due to redistribution) of a Ca2+-activatable Ca2+ channel has been recently reported only in blood neutrophils [7 11. An alternative to be considered is that the second messenger involved in SMOC regulation, although generated by receptor activation, is independent of both [Ca’+]i rise and PPI turnover. This possibility is supported by recent results with growth factors (GF). GF receptors are high-molecular-weight transmembrane proteins endowed with an endogenous protein kinase activity specific for tyrosine residues [81]. In addition, their activation causes PPI hydrolysis and [Ca2+]i to rise. Working with Swiss 3T3 cells we have observed that the contribution to this rise of the two components, redistribution and influx, varies greatly depending on the GF used. Platelet-derived GF and, especially, bombesin induce large PPI hydrolysis and redistribution responses, but fail to stimulate intlux to a detectable level; epidermal GF, on the other hand, induces only minor redistribution but marked influx. Thus, in contrast to redistribution, which remains correlated to PPI hydrolysis, influx does not, and could therefore be governed by separate signal(s) [82]. A similar explanation could account for the recent demonstration that pretreatment of adrenal granulosa cells with pertussis toxin blocks the infhrx of Ca”, but not PPI turnover and Ca2+ redistribution induced by vasopressin [83]. Taking all these results together it appears safe to conclude that, if indeed one (or more) second messenger operates SMOCs (and, in addition, justifies the name we propose for this group of channels), its nature remains at the best conjectural at the present time. Experiments with trappable [Ca2+]i indicators in a variety of cells exposed to appropriate receptor agonists while bathed in incubation media with or without Ca2+ (in order to compare the responses due to redistribution plus infhtx and

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redistribution only, respectively) have yielded consistent results. The observed [Ca2+]i rises were slightly different during the initial 30-60 s, but diverged later, being more persistent in the Ca2+-containing media. Based on these initial observations, the SMOCs have recently been proposed to be channels spanning not only the plasma membrane, but also the limiting membrane of the intracellular microsomal store [84] (Fig. 2). This hypothesis implies that a population of cistemae, previously reported in many cells to lie closely adjacent to the plasma membrane, is indeed coupled to it by SMOCs, giving rise to structural arrangements similar, at least functionally, to that of gap junctions. Opening of the SMOCs (possibly regulated by [Ca2’]i within the cisternae) would cause the refilling of this microsomal store, to balance the drainage by IP3-operated release [84]. At the present time support of this hypothesis is almost entirely speculative. Whatever it is, the mechanism that regulates SMOC activation must account for at least two features of the receptor-triggered [Ca’+]i responses: (1) the coupling of the two componentswith the partial exception of epidermal GF [82] mentioned above (which could indeed be a separate case), no conditions have been found in which influx is maintained and redistribution is blocked; and (2) the rapidity of the redistribution responses-redistributive [Ca’+]i rises have been measured within fractions of seconds from agonist application. Such a rapidity is explained if IP3-binding sites are located in close proximity of the plasmalemma, whereas it would be hard to account for if the sites were located (as it has often been assumed 14, 771) in the rough-surfaced endoplasmic reticulum, a structure that extends primarily into the deep cytoplasm. In addition, it should be mentioned that the number of IP3-binding sites, recently determined in hepatocytes, is relatively small [76] as would be expected for a location in a small membranebounded compartment, e.g., subplasmalemma cistemae, rather than in the very large endoplasmic reticulum; and that the size of the IP3-sensitive Ca” pool appears similar in various cell types, irrespective of the large variability of their endoplasmic reticulum. CONCLUSIONS The extremely asymmetrical distribution of CaZC across the plasma membrane offers the cells ample opportunity for the generation of both electrical and chemical intracellular signals (Fig. 2). The various Ca2+ channels that we have briefly described represent the tools used by cells for these purposes. Excitable cells, in particular neurons and neurosecretory cells, appear particularly well equipped, as they often express channels of all types The two existing mechanisms of channel activation, by voltage and chemical ligands, do not yield completely different effects. The various Ca 2+ VOCs generate electrical signals. In addition, activation of L (and, probably, also N) channels causes [Ca2’]i to rise quickly. Persistent cell depolarization causes a fraction of L channels to remain open and therefore causes [Ca’+]i to remain elevated for times (up to tens of minutes) that were unsuspected until recently [30]. ROCs sensu strictu resemble VOCs in structure and also in the rapidity of their effects. VOCs and ROCs are the channels that can cause the building up of steep intracellular [Ca2+]i gradients,

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with maxima beneath the plasma membrane, that classical as well as recent studies implicate in important functions, such as the stimulation of phasic, synchronous transmitter release at synapses and neurosecretory cells [3-5, 30, 85, 871. An additional feature of VOCs, whose extent remains to be fully established, but which is already of great interest, is their heterogeneous distribution at the surface of cells. For example, the proposed preferential localization of N channels in the presynaptic membrane [ 151 could account for the relative insensitivity of the process of neurotransmitter relase to DHP channel blockers [ 15, 16, 881. The role of SMOCs appears different from that of VOCs and ROCs. The function of these channels is intimately connected with the other process triggered by PPI-coupled receptor activation, i.e., Ca2+ redistribution, and consists primarily in the prolongation of the [Ca2+]i increases induced by that mechanism. Thus SMOCs appear involved not only in rapid cell activation, but also in the generation of slow-developing processes, such as cell growth. However, SMOCs and VOCs can sometimes work together with synergistic effects. For example, we have recently found that in rat mammotroph cells the [Ca2+]i response induced by thyrotropin-releasing hormone includes an initial spike, due mostly to redistribution, followed by a plateau contributed by both VOCs and SMOCs (unpublished results). Interactions between electrical and chemical signals occur also at VOCs of all types, which are variously modulated by second messenger-induced events (phosphorylations and, possibly, other events as well). Finally, it should be emphasized that many of the intracellular effects of Ca2+ are not induced by this messenger alone but are the result of interactions with other processes regulated by different second messengers. A good example is the regulation of secretion by exocytosis, which in a variety of cell types has been found to be governed by Ca2+ together with CAMP and protein kinase C-mediated events [42, 62, 74, 78, 89-911. Interaction among second messenger-operated events occurs therefore at multiple levels, constituting a complex network that encompasses whole physiological processes, from the sites of transduction of external stimuli to the final, effector responses. We thank our colleagues E. Wanke, F. Di Virgilio, D. Milani, L. M. Vicentini, A. Malgaroli, and A. Ambrosini for their contribution to the original work reported in this paper. Supported in part by grants from the CNR Special Project Oncology and the Italian Ministry of Education (40%). Note odded in proof Evidence accumulated during the last few months (reviewed in Ref. 92) suggests that in different cell types Ca*+ SMOCs might be activated by different intracellular second messengers (Ca *+ , IP4 and even IP3). It might be too early to draw final conclusions about these studies, in particular about the relative importance, and distribution in the various cell types, of the channels responding to these, and possibly other, second messengers.

REFERENCES 1. Carafoli, E. (1982) in Membrane Transport of Calcium (Carafoli, E., Ed.), p. 109, Academic Press, London. 2. Blaustein, M. P. (1984) in Electrogenic ‘Bansport: Fundamental Principles and Physiological Implications (Blaustein, M. P., and Lieberman, M., Eds.). p. 129, Raven Press, New York.

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3. Baker, P. F. (1986) in Ion Channels in Neuronal Membranes (Ritchie, M. J., Keynes, R. D., and Bolis, L., Eds.), p. 177, A. R. Liss, New York. 4. Berridge, M. J., and Irvine, R. (1984) Nature (London) 312, 315. 5. Sekar, M. C., and Hokin, L. E. (1986) J. Membr. Biol. 89, 193. 6. Rasmussen, H. (1986) N. Engl. .I. Med. 314, 1094. 7. Hag&at-a, P. G. (1981) Biochim. Biophys. Acta 650, 128. 8. Kostyuk, P. G. (1981) Biochim. Biophys. Acta 650, 128. 9. Janis, R. A., and Biggie, D. J. (1983) J. Med. Chem. 26, 775. 10. Hamill, O., Marty, A., Neher, E., Sackmann, B., and Sigworth, F. J. (1981) Pfiiger’s Arch 391, 85.

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24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

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Received October 10, 1986 Revised version received December 15, 1986

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