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E-Conference: Defining Calcium Entry Signals

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Comments on cellular domains that contribute to calcium entry events

4 June 2004

Michael Berridge

Comments on Question 2: What is known about the cellular domains contributing to the transduction of signals into Ca2+ entry events?

I shall comment on this question by re-interpreting my earlier conformational coupling hypothesis (Berridge 1995 Biochem. J. 312:1) in the light of new developments. The basic idea, as originally proposed by Robin Irvine, is that stimulation of the IP3 receptor (IP3R) induces a conformation change that is transferred to the channel in the plasma membrane through a direct protein-protein interaction (Irvine 1990 FEBS Letters263:5). The entry channels have yet to be defined and current contenders are ICRAC and some of the Trp channels. Just how this coupling complex is activated is a critical issue.

In the hypothesis I developed earlier, I proposed that the IP3R could be activated either by IP3 or by store depletion and both might be operating. Strong evidence for the former was provided recently in a study on DT40 cells that lack IP3Rs and fail to induce entry or release following agonist stimulation. However, Ca2+ entry was restored upon expression of an IP3R even when it had a C-terminal truncation preventing it from releasing Ca2+ (van Rossum et al 2004 PNAS 101: 2323). The IP3R was able to respond to IP3 and to induce the conformational change necessary to promote an entry of Ca2+ even though it was unable to release Ca2+ from the store. This observation is in line with another prediction of the original model that suggested that when the IP3R is coupled to the entry channel it is non-conducting and thus not able to release Ca2+.

It is the operation of this conformational coupling mechanism at physiological agonist concentrations that will be a primary focus of the comments outlined below that will be based on a series of questions:

  1. What are the Ca2+ entry signals in primary cells and does conformational coupling play any role as an entry mechanism?

Studies on cloned cell lines have identified a number of putative entry signals such as DAG, IP3, arachidonic acid, store emptying etc, but what is the evidence that any of these actually play a role in primary cells? Studies on various primary cells certainly indicate that agonist can induce Ca2+ entry and in some cases this has been linked to Trp channels, but the precise nature of the coupling mechanism is largely obscure. There are an increasing number of examples of agonist-induced entry:

  1. It Mast cells, receptor activation stimulates a Ca2+ release activated current (ICRAC) (Hoth & Penner 1992 Nature 355: 353).
  2. In Purkinje neurons, metabotropic glutamatergic receptors activate Ca2+ entry through TRPC1 channels apparently independently of IP3 (Kim et al 2003 Nature 426:285).
  3. In thalamic interneurons, 5-HT2 receptors activate TRPC4 receptors to induce Ca2+ entry that triggers the dendritic release of GABA (Munsch et al 2003 PNAS 100:16065) .
  4. Fertilization of mammalian oocytes results in the activation of Ca2+ entry responsible for driving Ca2+ oscillations (McGuiness et al 1996 Development 122:2199).
  5. In pontine neurons, BDNF stimulates Ca2+ entry through TRPC3 channels (Li et al 1999 Neuron 24:261).
  6. Information on additional examples of Ca2+ entry mechanisms in primary cells would be welcome.

It is clear from the above that primary cells may employ a number of different mechanisms for coupling receptor activation to Ca2+ entry that can occur independently of store depletion. However, as indicated in the conference perspective, there is a prevalent assumption that "Receptor-induced Ca2+ signals involve two closely coupled events: Ca2+ release from ER stores, and Ca2+ entry across the plasma membrane." The way in which release might be coupled to entry is through activation of store-operated channels (SOCs) as first proposed by Putney (1986 Cell Calcium 7:1). There is no doubt that such a store-operated mechanism is unveiled when stores are depleted using thapsigargin or high doses of agonist, but is there any evidence that such a mechanism exists following agonist stimulation at physiological doses?

Is it correct to assume that these two processes are always coupled or is it possible for agonists to activate entry independently of release? The answer to this question is probably yes and the model outlined earlier suggests one possible mechanism i.e. the conformational change in the IP3R responsible for stimulating the entry channel can be activated either by IP3 or by store depletion. While both mechanism can activate entry, it is possible that their primary functions are different: the direct IP3 activation pathway may be dedicated to coupling receptor activation to entry whereas the store-operated pathway may be a homeostatic mechanism that ensures the internal stores remain filled. Having such a dual activation mechanism may also explain how entry can be activated by receptors that generate IP3 or by mechanism that deplete stores independently of IP3 as occurs following activation of ryanodine receptors.

To fully understand how entry is regulated it is necessary to consider the critical question of the sensitivity of these entry mechanisms and how they are activated at physiological agonist concentrations.

  1. What is the dose response relationship for the activation of calcium entry signals relative to the downstream physiological responses?

Dose-response relationships have been largely ignored even though they are crucial in order to understand the physiological mechanisms of Ca2+ entry. I am not aware of any dose-response curves for the sequential events of agonist-induced IP3 formation, Ca2+ entry and subsequent downstream cellular responses. I attempted to do this with the insect salivary gland by measuring the EC50 values for 5-HT induced fluid secretion (3x10-9M), the transepithelial flux of Ca2+ as an indirect measure of Ca2+ entry (1.5 x 10-8 M) and inositol efflux to monitor IP3 formation (5x10-7 M) (Fain and Berridge 1979 Biochem. J. 178:45). The curves for Ca2+ entry and IP3 formation were displaced to the right of the physiological response such that a 50% activation of secretion was seen at a 15% activation of entry and a 2.5% activation of IP3 formation. The point of this example is to stress that a very small activation of IP3 formation is sufficient to induce the small activation of Ca2+ entry necessary to fully activate a downstream physiological response. A similar conclusion emerges if one considers the agonist concentrations responsible for setting up Ca2+ oscillations.

C. What is the role of Ca2+ entry signals in driving Ca2+ oscillations?

For some cell types, the low agonist concentrations that activate physiological responses also induce Ca2+ oscillations. It is often assumed that Ca2+ entry signals and, in particular the SOCs, function to maintain such oscillations by recharging the internal store following each spike. It is relevant to ask, therefore, how much of the store is released during the course of a spike and what is the level of Ca2+ entry? When the ER lumenal level of Ca2+ was monitored in a pancreatic cell spiking regularly in response to a low dose of acetylcholine, it declined by about 5% during each spike (Park et al (2000) EMBO J. 19:5729). This small loss was then gradually replenished during the next interspike interval. In many cells, this refilling of the partially depleted store occurs during the course of the interspike interval even though the cytosolic level of Ca2+ is close to its resting level. This implies that the rate of Ca2+ entry is very low and is rapidly taken up by the ER to set the stage for the next spike. Indeed, I consider that it is the rate of Ca2+ entry that is the timing mechanism for the frequency-modulated (FM) signaling mechanism seen in many cell types.

As the entry of Ca2+ necessary to maintain oscillations occurs with a minimal depletion of the internal store this would seem to rule out a role for a store-operated signalling mechanism. However, this may not be the case as will be discussed in the following section.

  1. Is there a role for conformational coupling mechanisms in regulating entry at physiological agonist concentrations?

As Ca2+ entry in many cells (Section A) is driven by receptors that stimulate phospholipase C, the most likely entry signals are either IP3 or DAG or a related lipid intermediate. Since DAG has been shown to activate certain Trp channels, it cannot be ruled out. However, it would seem that IP3-induced entry is a more likely mechanism because the introduction of IP3 into the cell can activate Ca2+ signaling including the more complex patterns of oscillations that depend on entry. Since there is no evidence for IP3 activating entry channels directly (except in some sensory cells), it is argued that it acts through the conformational coupling mechanism outlined earlier.

E. Conformational coupling- a revised hypothesis

In order to understand how the conformational coupling mechanism might operate to regulate entry, especially at low agonist concentrations, it is necessary to consider the structural organization of the coupling unit within the context of the tubular ER network. As proposed previously (Berridge,1995), small fingers of this network form flattened sacks that make close contact with the plasma membrane to form the specialized junctional zones where the conformational coupling units are located. The10 nM gap separating the ER sacks from the plasma membrane has periodic densities that are thought to be the large cytoplasmic heads of the IP3R, which communicate information to the entry channels. These junctional zones appear to be few in numbers, which may explain why the rate of entry at physiological concentrations is so low.

An important issue arises as to whether these junctional zones are coupled to the bulk of the ER as is assumed in this hypothesis or whether there is a separate store associated with the plasma membrane that is dedicated to controlling entry as some have suggested. My argument against the latter mechanism is that it is inconsistent with the homeostatic function of store-operated entry, which is to ensure that the store remains filled. I would argue that a small store may fill up quickly and entry would cease even when the bulk of the ER is empty and requires further Ca2+.

As described in Section B the formation of IP3 at physiological agonist concentrations is very low raising the possibility that it occurs it is punctate i.e. IP3 may appear as brief highly localized pulses in small domains beneath the plasma membrane. If these localized domains of IP3 coincide with a region where a junctional zone is located, conformational coupling will be activated. This localized activation could be mediated by IP3 in two ways. Firstly, IP3 could act directly to stimulate the IP3Rs that are coupled to the entry channels. Secondly, it could act indirectly by stimulating uncoupled IP3Rs in the vicinity of the junctional zone to induce a localized depletion of the store within the ER to switch on a store-operated mechanism. This latter case could explain how a store-operated mechanism could operate to regulate entry during the low agonist conditions that induce Ca2+ oscillations. The highly localized depletion in the junctional zones control entry while the bulk of the Ca2+ is kept within the main body of the ER where it is available for the generation of Ca2+ spikes.

Whatever the activation mechanism turns out to be, the critical point is that entry is driven by a conformational coupling mechanism restricted to the junctional zones that are responsible for bringing Ca2+ into the cell prior to the onset of the Ca2+ spike. In the case of the insect salivary gland, I obtained some indirect evidence that 5-HT might be capable of stimulating entry during the latent period before the first spike occurred (Berridge 1994 Biochem J. 302:545). At low agonist concentrations, the earliest Ca2+ response to receptor activation may be the stimulation of entry, which is then responsible for charging up the internal store to prime the Ins3Rs for the large-scale regenerative release of Ca2+ that occurs during each spike.

Is it possible that the long held assumption the entry follows release might in fact be quite the opposite? Perhaps the earliest Ca2+ response at physiological agonist concentrations is an increase in entry that then sets the stage for Ca2+ release.

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