The calcium store in the nuclear envelope

Cell Calcium (1998) 23(2/3), 87-90 0 Harcourl Brace & Co. Ltd 1998

Invited review

The calcium store nuclear envelope Ole H. Petersen, Oleg V. Gerasimenko, Hideo Mogami, Alexei V. Tepikin MRC Secretory

Control

Research

Group,

Physiological

Laboratory,

in the Julia V. Gerasimenko,

University

of Liverpool,

Liverpool,

UK

Summary The nuclear envelope has a relatively small volume, but is connected up to the vastly larger endoplasmic reticulum. The Ca*+ concentration in the lumen of the interconnected nuclear envelope and endoplasmic reticulum network is in the resting state maintained at a level of more than 100 PM. There are specific Ca2+ release channels present in the inner nuclear membrane that can be activated by inositol trisphosphate or cADP ribose. The system, therefore, allows selective release of Caz+ into the nucleoplasm which could be important for the control of specific types of gene expression.

CONNECTIVITY OF THE LUMEN NUCLEAR ENVELOPE AND THE ENDOPLASMIC RETICULUM

OF THE

NON-UNIFORM IN VARIOUS

It is generally accepted that the outer nuclear membrane (ONM) has endoplasmic reticulum (ER) characteristics and is continuous with the ER membranes, whereas the protein composition of the inner nuclear membrane (INM) is quite different from that of the ONM [l]. Anatomically, the lumen of the nuclear envelope (NE) is in direct continuity with the ER lumen [l], but this does not necessarily mean that there is rapid diffusion of Ca2+ and other molecules between the lumen of the ER and the NE. This important question has been addressed very directly in a recent study by Subramanian and Meyer 121. They used a lumenal elastase-GFP fusion protein and showed that this large molecule could rapidly diffuse across the cell within the lumenal storage space defined by the ER and NE [2]. Since there is evidence for movement of Ca2+ within the ER lumen over large distances from one end of a cell to the other [3], we can envisage a combined ER-NE Ca2+ store with a uniform resting Ca2+concentration.

The operation of the ER-NE as one luminally continuous Ca*+ store does not necessarily imply uniform transport properties throughout the cell. Studies on polarized pancreatic acinar cells clearly show that Ca2+release channels can be clustered in particular areas. The primary inositol trisphosphate (IPJ-induced Ca*+ release sites in pancreatic acinar cells are localized in the apical secretory granule containing region [4,5] and immunolocalization studies show that all the three subtypes of the IP, receptor are found exclusively in the apical part of the cell close to the luminal and lateral membranes [6]. The distribution of the IP, receptors correlates very well with the primary Ca2+ release sites [3,6,7]. The whole of the NE-ER Ca2+store can be utilized to provide Ca2+for local release in the region of the secretory granules which is of course the strategically important area for secretion control [3]. In such a widely connected Ca2+ store one can predict a uniform resting Ca2+ level throughout the store, but transiently there could be considerable local Ca2+ gradients within the ERNE system. DISTRIBUTION AND PUMPS

Correspondence to: Prof. O.H. Petersen, MRC Secretory Control Group, The Physiological Laboratory, The University of Liverpool, Street,

Liverpool

L69 3BX, UK.

Fax:

+44

151

794

5323

Research Crown

Ca*+ TRANSPORT PROPERTIES PARTS OF THE ER-NE STORE

OF Ca*+ RELEASE IN THE NE

CHANNELS

In the model proposed by Baths et al. [8], it was envisaged that both IP, receptors and Ca2+-ATPaseswere

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present in both the INM and the ONM. Recent studies have given direct information about the specific localization of transport proteins in the NE. In studies on isolated mouse liver nuclei, Gerasimenko et al. [9] showed that IP, could evoke Ca2+ release from the NE that was primarily directed into the nucleoplasmic space, indicating that the functionally important IP, receptors were in the INM. Furthermore, Hennager et al. [lo] showed in Xenopus oocytes that IP, injected into the nucleoplasm could elevate the nucleoplasmic Ca2+ concentration. This effect could be blocked by nucleoplasmic, but not cytoplasmic, heparin application. In a biochemical study in which it was possible to separate INM and ONM from rat liver nuclei, it was shown that the high affinity IP, receptors were selectively localized in the INM, whereas the Ca2+-ATPase was exclusively found in the ONM [ 1 I]. More recently, Santella and Kyozuka [ 121 have shown in experiments on starfish oocytes that the IP,-induced rise in nucleoplasmic Ca2+ concentration is blocked not only by an intranucleoplasmic heparin injection but also by injecting into the nucleoplasm an antibody against the IP, receptor. Overall, the evidence for the existence of functionally important IP, receptors in the INM is very strong [9-121. In contrast, the Ca2+-ATPase,which is identical to the one in the ER [ 131, is exclusively present in the ONM [l l] (Fig. 1). From patch clamp studies, there is also evidence for functional IP, receptors in the ONM [ 14,151, but it is perhaps not absolutely clear whether the presence of ER adhering to the isolated nuclei can be excluded with certainty in these experiments. Patch clamp recording of small ion channels in the ONM would, in principle, be expected to be very difficult in view of the high density of such large channels as the nuclear pore complexes (NPC). In addition to the IP, receptors, there are also receptors in the INM for the putative Ca2+ releasing messenger cADP ribose (cADPr). Gerasimenko et al. [9] showed that cADPr added to isolated liver nuclei evoked release of CaZ+ from the NE that was primarily directed into the nucleoplasm indicating the presence of functional cADPr (ryanodine) receptors in the INM. More recently, Santella and Kyozuka [ 121 have shown that, in starfish oocytes, injection of cADPr into the nucleoplasm resulted in a nucleoplasmic Ca2+ concentration rise which was abolished by injection of the cADPr receptor antagonist &amino cADPr into the nucleoplasm, as well as by injection of an antibody against ryanodine receptors. Photo release of caged cADPr in the nucleoplasm gave rise to repetitive nucleoplasmic Ca2+ spikes [ 1.21.These data [9,12] clearly indicate the presence of cADPr activatable Ca2+ release channels, most likely belonging to the ryanodine receptor family, in the INM (Fig. 1). Cell Calcium

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03NPC +

Ca’+ATPase

Nucleoplasm

Fig. 1 Diagram showing Ca2+ transport pathways in the nuclear envelope (NE) and the endoplasmic reticulum (ER). ONM, outer nuclear membrane; INM, inner nuclear membrane; NPC, nuclear pore complex.

IS THE

NPC

PERMEABLE

TO Ca*+?

The selective presence of Ca2+ release channels in the INM combined with the selective presence of the Ca2+ ATPase in the ONM (Fig. 1) immediately presents a problem. There seems to be a mechanism for selectively moving Ca2+ into the nucleoplasm, but how does the nucleoplasm dispose of this Ca2+?In studies on isolated liver nuclei, Gerasimenko et al. [9] showed that the IP,or cADPr-evoked nucleoplasmic Ca2+ rise was only transient. It was also shown that acute changes in the Ca2+ concentration outside the isolated nuclei were quickly reproduced in the nucleoplasm. The conclusion was, therefore, that Ca2+ released through the INM into the nucleoplasm could diffuse into the cytosol via the permeable NPCs [9]. There is ample evidence also in intact cells showing that Ca2+ readily diffuses through the NPCs. Although some studies appear to indicate that Ca2+does not move easily through NPCs, the elegant and thorough analysis made by Lipp et al [ 161 convincingly shows that Ca2+does actually diffuse rapidly through the NPCs. This does not mean that the cytosolic and nucleoplasmic Ca2+ concentrations are always identical in all cells. Local cytosolic Ca2+ signals can be generated that simply do not reach the NE [ 171. In the pancreatic acinar cells, local cytosolic Ca2+ signals in the apical secretory granule area can be substantial and cytosolic Ca2+ gradients of up to 400 nM/l.tm can be established transiently along the line connecting the secretory pole with the nucleus [17]. It is entirely possible that local nucleoplasmic Ca 2+ transients generated by opening of Ca2+ release channels in the INM would not be transmitted to the cytosol, but rather disappear in the cytosol due to dilution and Ca2+ uptake into the densely packed ER. 0 Harcourt

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CP in nuclear envelope

THE

CaZ+ CONCENTRATION

IN THE

NE STORE

In studies on isolated liver nuclei, an ATP-dependent Ca*+ uptake into the NE store could be demonstrated [9]. In these experiments, the high-affinity Ca2+-sensitive fluorescent probe Fura- was used. With hindsight, it is clear that this was a less than ideal choice. One would expect the resting Ca2+concentration inside the NE to be relatively high and it would, therefore, have been better to employ a low affinity probe such as, for example, MagFura-2. When this was done later [Gerasimenko, Gerasimenko & Tepikin, unpublished] the result indicated that in the isolated liver nuclei, the Ca2+-pump mediated Ca2+ uptake only lead to Ca2+ concentrations inside the NE store of up to a few micromoles per litre. In this context, one should bear in mind that in the process of isolating nuclei, connections with the ER are broken and artificial leaks in the ONM are, therefore, inevitable. To what extent resealing occurs is unknown, but it seems not unreasonable to suspect that the resealing process is less than perfect. If there is an unphysiologically high leak, the system may not be able to reach the normal steady state level. In order to obtain relevant information about the normal Ca2+concentration in the lumen of the NE ([Ca2+]J it may, therefore, be better to attempt an estimation of [Ca2+], in the ER of intact cells. We have recently made measurements on intact pancreatic acinar cells which have been loaded with Mag-Fura-2. This probe has subsequently been washed out of the cell into a patch pipette (whole cell recording configuration) so that only probe molecules retained inside the ER-NE lumen are of quantitative importance. With this technique, resting [Ca2+lLvalues of 100-300 l.tM have been obtained [ 181. In experiments where Ca2+ re-accumulation into the store following agonist-evoked release was monitored, it was observed that the apparent uptake rate decreased steeply with an increase in [Ca2+], (negative feedback inside ER lumen). Leak rates were assessed by application of thapsigargin, the selective inhibitor of the Ca2+-ATPase pumps in the store membrane. Above [Ca2+lLof 100 m, the Ca2+ leak was essentially independent of [Caz+lL.The steady state resting [Ca”], represents the equilibrium point at which leak and uptake balance exactly and this seems to occur at [Ca2+], values of 250-350 @VI.The main conclusion from this work is that the negative feedback of Ca2+inside the NE-ER lumen on the Ca2+pump effectively controls the Ca2+store content [ 181. THE

FUNCTION

OF THE

NE Ca*+ STORE

It is the presence of Ca2+release channels in the INM that makes the NE Ca2+ store functionally important; otherwise it would just be an insignificant part of the ER. Although the volume of the NE is very small, it is 0 Harcouti

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Fig. 2 Very schematic illustration of Ca*+ movement across cell membrane, ER membranes and nuclear envelope (NE) membranes. SOC, store-operated Ca*+ channel; PM cell membrane; ER, endoplasmic reticulum.

connected up to the vastly larger ER store and, because of the mobility of Ca2+ inside the store and the luminal continuity of the whole NE-ER complex [2,3,18], Ca2+can in principle be effectively mobilized into the nucleoplasm (Fig. 2). In this context, it is very important that the whole apparatus for IP, production is present inside the nucleus [ 191. The recent demonstration of the distinct functions of nuclear and cytoplasmic Ca2+ in the control of gene expression 1201 makes it very important to search for possible specific nuclear Ca2+signals. Figure 2 summarizes the Ca2+ transport pathways that are of particular importance for the nucleus. The extracellular solution and the inside of the NE-ER network are two compartments with a high Ca2+ concentration (> 100 @I) whereas the cytosol and the nucleoplasm are low Ca2+ concentration compartments with a Ca2+ concentration of around 100 nM at rest. Locally, following stimulation, the Ca2+ concentration close to clusters of IP, or cADPr receptors may rise transiently to a few @VI. Ca2+ signals generated in the nucleoplasm close to the Ca2+ release channels in the INM may quickly be dissipated due to diffusion of Ca2+ through the permeable NPCs and subsequent dilution in the cytoplasm as well as uptake into the ER. Any rise in the cytosolic Ca2+ concentration will activate Ca2+ pumps in the plasma membrane [21] extruding Ca2+ into the extracellular solution. Depletion of Ca2+ in the NE-ER Ca2+ store will open store-operated Ca2+ channels in the Cell Calcium

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plasma membrane allowing Caz+ entry [22]. This process does not necessarily cause any change in the cytosolic Ca2+concentration because of rapid local uptake into the ER [3,18].

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