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Advan. Physiol. Edu. 33: 169-174, 2009; doi:10.1152/advan.00025.2009
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ADV PHYSIOL EDUC 33:169-174, 2009
© 2009 American Physiological Society

RESEARCH-ARTICLE

Teaching the modes of Ca2+ transport between the plasma membrane and endoplasmic reticulum using a classic paper by Kwan et al.

Willmann Liang

School of Biological Sciences, Nanyang Technological University, Singapore

Address for reprint requests and other correspondence: W. Liang, School of Biological Sciences, Nanyang Technological Univ., Singapore 637551 (e-mail: willmann{at}ntu.edu.sg).

Abstract

This teaching article uses the report by Kwan et al., "Effects of methacholine, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells," where the effects of Ca2+-mobilizing agents in regulating Ca2+ fluxes were examined under various conditions. Upper-level undergraduate and new graduate students in physiology are the targe audience. Teaching and learning points are put forth in this article to illustrate 1) the characteristics of methacholine- and thapsigargin-induced Ca2+ responses, 2) the different endoplasmic reticulum Ca2+ stores accessible to methacholine and thapsigargin, 3) the inhibitory effects of La3+ on Ca2+ extrusion and Ca2+ influx, and 4) the facilitatory role of La3+ on endoplasmic reticulum Ca2+ recycling. Each of the above concepts is first explained with references to the figures adapted from the original article. A list of student learning questions then follows, where the answers are found in the teaching notes for the instructors. It is the objective of this article to make both teaching and learning Ca2+ regulation a rewarding experience for all.

Key words: Ca2+ recycling; capacitative Ca2+ entry; education

THE IMPORTANT ROLES of Ca2+ in cell physiology cannot be emphasized more. In neuronal endings, Ca2+ entry triggers the vesicular release of neurotransmitters. In cardiac and smooth muscle, the coupling between Ca2+ influx and release governs contractility. In endothelial cells, Ca2+ mobilization precedes the synthesis and secretion of various vasoactive substances. A specific mode of Ca2+ influx, capacitative Ca2+ entry (CCE), is particularly important in nonexcitable cells, of which endothelial cells are an example. Other terms for CCE include store-operated and store-mediated Ca2+ entry (9). The prevailing significance of CCE in nonexcitable cells may be explained by the lack of functional voltage-gated Ca2+ entry in these cells. In such cells where electrical stimulation (via membrane depolarization) yields minimal or no Ca2+ movement, CCE provides the means to refill depleted intracellular Ca2+ stores.

It is currently accepted that depletion of the endoplasmic reticulum (ER) Ca2+ store brings about the redistribution of a Ca2+ sensor protein, stromal interacting molecule-1 (Stim1) from within the ER to areas closer to the plasma membrane (PM) (8). Without leaving the ER, Stim1 may interact with the PM protein Orai1, which is in close proximity with the ER Ca2+ sensor protein (5, 8). A diffusible Ca2+ influx factor, as proposed by Bolotina and Csutora (1), may also be involved in the activation of Orai1. Orai1 contains subunits that form pores on the PM that allow Ca2+ passage (6). The resulting Ca2+ current is commonly termed Ca2+ release-activated Ca2+ current (3) and forms the basis of CCE that was observed by Putney in 1986 (7).

Compelling evidence of the modes of Ca2+ flux in CCE was presented by Kwan et al. in an APS classic paper in 1990 (4). The stated article has been cited 188 times as of March 2009 and is the subject of study in this teaching article, which is aimed at upper-level undergraduate and new graduate students in physiology. Although not a focus of the original article, the basic principles of cytosolic Ca2+ measurements warrant a brief introduction here. Kwan et al. (4) loaded lacrimal acinar cells with fura-2 and captured fluorescence signals with a spectrofluorimeter. Two wavelengths, 340 and 380 nm, are used alternately to excite fura-2-loaded cells typically. When fura-2 is unbound or Ca2+ free, fluorescence is emitted at 340-nm excitation. On the other hand, when fura-2 is bound by Ca2+, fluorescence is emitted at 380-nm excitation. The emitted fluorescence is captured at 500 nm. The fluorescence ratio between 340 and 380 nm is calculated and inserted into an equation described by Grynkiewicz et al. (2). Values of absolute Ca2+ concentration are determined using this equation. To make use of the equation, minimal and maximal fluorescence ratios are also needed, and these can be obtained by permeabilizing the cells and subjecting them to high Ca2+ with and without a chelator. The measurement of fluorescence ratios in determining relative Ca2+ concentration earns fura-2 the description of a ratiometric Ca2+ indicator. Another ratiometric dye commonly used is indo-1. Only one excitation wavelength (at 338 nm) is used with indo-1, but fluorescence signals are captured at two emission wavelengths, usually 405 and 485 nm. Ca2+-free indo-1 emits fluorescence at 485 nm, whereas the Ca2+-bound dye emits at 405 nm. Since leakage of dyes does not affect the calculated fluorescence ratios, ratiometric Ca2+ indicators remain very useful tools in cytosolic Ca2+ measurements.

Using Ca2+-mobilizing agents on lacrimal acinar cells, Kwan et al. demonstrated that ER Ca2+ refilling after store depletion occurs via a two-step process (4). First, extracellular Ca2+ enters the cytosol or, alternatively, Ca2+ released from the ER is retained in the cytosol. Cytosolic Ca2+ is then taken up by the ER to refill the Ca2+ store. Using the article by Kwan et al. (4) as a reference, I will propose some teaching and learning points on drug-induced Ca2+ responses, ER Ca2+ recycling, and CCE in the following areas: 1) methacholine (MeCh)-induced ER Ca2+ release and Ca2+ influx, 2) thapsigargin (TSG)-induced ER Ca2+ release and Ca2+ influx, 3) differences in ER Ca2+ stores activated by MeCh and TSG, 4) dual effects of La3+ in blocking Ca2+ extrusion and Ca2+ influx, and 5) facilitation of ER Ca2+ recycling by La3+.

MeCh-Induced ER Ca2+ Release and Ca2+ Influx

Teaching points for student handout 1. Figure 1A (adapted from Fig. 1A of the original article) compares Ca2+ responses elicited by 3 µM MeCh in different extracellular Ca2+ environments. Trace 1 shows a biphasic MeCh-induced Ca2+ response with a sustained plateau after the initial upstroke. The plateau was maintained by continuous Ca2+ influx as 1 mM Ca2+ was present in the bathing solution. Upon repeated additions of the Ca2+ chelator EGTA (at points a and b; Fig. 1A), Ca2+ influx was diminished and eventually abolished. Trace 2 in Fig. 1A shows a MeCh-induced Ca2+ response obtained in nominal Ca2+-free solution. No Ca2+ was added to the bathing solution in this case. In addition, 3 mM EGTA was added (at point c; Fig. 1A) to chelate any trace amounts of Ca2+ that may be present. The result was a transient MeCh-induced Ca2+ response with no plateau phase, i.e., no Ca2+ influx. A comparison between traces 1 and 2 reveals that the initial upstroke and decay phase of the Ca2+ response is contributed by ER Ca2+ release, independent of extracellular Ca2+. Trace 3 in Fig. 1A was obtained much like trace 2 except that EGTA was not added and 1 mM Ca2+ was added (at point d; Fig. 1A) to the bathing solution at the end of the transient Ca2+ response. This elicited a Ca2+ plateau to a level similar to that seen in trace 1 (before EGTA was added). A comparison between traces 1 and 3 indicates that the processes of ER Ca2+ release and Ca2+ influx can be functionally separated by manipulating the extracellular Ca2+ environment. Trace 3 in Fig. 1A also demonstrates an example of CCE when the MeCh-sensitive ER Ca2+ store is depleted.


Figure 1
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  		Fig. 1. Methacholine (MeCh)-induced Ca2+ release and Ca2+ influx. A: MeCh-elicited Ca2+ responses with (trace 1) and without (traces 2 and 3) extracellular Ca2+. B: abolition of the MeCh-elicited Ca2+ response with the addition of drug at the indicated arrows. See the teaching notes for the identity of substances added at points a-d and at the indicated arrows.

 
Figure 1B (adapted from Fig. 1B of the original article) shows that the Ca2+ response elicited by MeCh was due to the activation of muscarinic receptors. A prior incubation with atropine (a muscarinic antagonist) prevented MeCh-induced Ca2+ mobilization (trace 1; Fig. 1B). The addition of atropine during the plateau phase of the MeCh-induced Ca2+ response abolished the response completely (trace 2; Fig. 1B).

Student handout 1. Figure 1 is included in the handout.

  1. Fill in the blanks (at points a-d) to indicate whether EGTA or Ca2+ was added in traces 1–3 in Fig. 1A.
  2. Was extracellular Ca2+ present during the course of traces 1 and 2? How were the responses different and why?
  3. In Fig. 1B, the addition of which drug (at points indicated by arrows) would have resulted in the responses seen in traces 1 and 2?

TSG-Induced ER Ca2+ Release and Ca2+ Influx

Teaching points for student handout 2. Figure 2A (adapted from Fig. 3A of the original article) shows the relationship between TSG concentration and the resultant Ca2+ responses in nominal Ca2+-free solution followed by the addition of 3 mM Ca2+ to the bathing solution. By inhibiting ER Ca2+-ATPase so that cytosolic Ca2+ cannot be taken up into the ER, TSG causes a gradual depletion of the ER Ca2+ store. High TSG concentrations (0.7 and 1 µM) induced ER Ca2+ release to a similar extent (traces 1 and 2; Fig. 2A) with nearly equal time courses. The subsequent addition of 3 mM Ca2+ induced Ca2+ influx, as indicated by the sustained plateau phase. The submaximal TSG concentration of 0.3 µM elicited a slightly smaller maximal Ca2+ response with a longer time course (trace 3; Fig. 2A) and was followed by a plateau of similar amplitude as traces 1 and 2 in the presence of extracellular Ca2+. At 0.03 µM, TSG elicited a much slower Ca2+ response (trace 4; Fig. 2A). The maximum attained Ca2+ concentration [intracellular Ca2+ concentration ([Ca2+]i)] was also lower than those in traces 1–3 but the subsequent plateau was higher. However, when taken from the net difference between the sustained plateau and end level of the preceding response from ER Ca2+ release (i.e., between points i and ii on traces 1–3 and points iii and iv on trace 4), the plateau was essentially of the same amplitude regardless of TSG concentration. Altogether, from traces 1–4, it can be concluded that TSG causes the ER Ca2+ store to deplete completely, which, in the presence of extracellular Ca2+, triggers CCE. Trace 5 in Fig. 2A shows marginal TSG-induced ER Ca2+ release and Ca2+ influx only due to the very low TSG concentration (0.01 µM) used.


Figure 2
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  		Fig. 2. Thapsigargin (TSG)-induced Ca2+ release and Ca2+ influx. A: TSG-elicited Ca2+ responses in nominal Ca2+-free solution. The range of TSG concentrations used was from 0.01 to 1 µM, which corresponds with traces 2-6. No TSG was present in trace 1. B: effects of varying extracellular Ca2+ concentrations on TSG responses. The Ca2+ concentrations used were as follows: 0.3 mM in trace 3, 1 mM in trace 4, and 3 mM in trace 5. No Ca2+ was present in traces 1 and 2. EGTA was present in trace 1 only. See the teaching notes for the identity of substances added at points a–c, points i-iv, and the individual TSG concentrations used in each trace.

 

Figure 3
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  		Fig. 3. Endoplasmic reticulum (ER) Ca2+ stores accessible to MeCh and TSG. A: MeCh-elicited Ca2+ responses were followed by the application of varying TSG concentrations in nominal Ca2+-free solution. No TSG was present in trace 4. Extracellular Ca2+ was added to elicit the final Ca2+ response. B: Ca2+ responses elicited by varying TSG concentrations were followed by MeCh application. Extracellular Ca2+ was added to elicit the final Ca2+ response. C: inositol 1,4,5-trisphosphate (IP3)-induced ER Ca2+ release was followed by the application of TSG. Heparin antagonizes the IP3 receptor and blocks Ca2+ release. D: application of IP3 after TSG failed to elicit a Ca2+ response. See the teaching notes for the individual TSG concentrations used and the corresponding traces.

 
Also shown in Fig. 2A is that the effective CCE was stimulated by 0.03-1 µM TSG. Figure 2B (adapted from Fig. 3B of the original article) compares 0.3 µM TSG-elicited Ca2+ responses in varying extracellular Ca2+ concentrations. The increase in extracellular Ca2+ concentration from 0 to 3 mM was reflected by the higher amplitudes of the TSG-induced sustained Ca2+ plateau in the various traces [with extracellular Ca2+ concentrations: trace 1 (0 mM + EGTA), trace 2 (0 mM), trace 3 (0.3 mM), trace 4 (1 mM), and trace 5 (3 mM); Fig. 2B]. The amount of Ca2+ influx is thus dependent on extracellular Ca2+ concentration. For trace 2, where extracellular Ca2+ was absent until the addition of 3 mM Ca2+, the resultant Ca2+ plateau reached a similar level to that of trace 5, the extracellular Ca2+ concentration of which was 3 mM throughout. This serves as further evidence of the importance of extracellular Ca2+ concentration in regulating the amount of Ca2+ influx after ER Ca2+ store depletion. Thus, if 3 mM Ca2+ is added in traces 3 and 4 (at point a; Fig. 2B), the subsequent plateau would also reach a similar level to that of trace 5. For trace 1, no plateau would be expected even if 3 mM Ca2+ is added since the bathing solution contains EGTA also. The addition of EGTA in trace 5 abolished the Ca2+ plateau, again demonstrating the source of Ca2+ being extracellular during the plateau phase.

Student handout 2. Figure 2 is included in the handout.

  1. Fill in the blanks (at points a–c) to indicate whether EGTA or Ca2+ was added in Fig. 2, A and B.
  2. Rank the order of TSG concentration (0, 0.01, 0.03, 0.3, 0.7, and 1 µM) added in traces 1–6 in Fig. 2A.
  3. In Fig. 2A, trace 4 shows a TSG-elicited Ca2+ response that is smaller and slower than those in traces 1–3. Did complete ER Ca2+ depletion occur? Justify your answer.
  4. In Fig. 2B, predict the level of Ca2+ influx for traces 1, 3, and 4 if the extracellular Ca2+ concentration is increased to 3 mM at point c.

Differences in ER Ca2+ Stores Activated by MeCh and TSG

Teaching points for student handout 3. Figure 3 (adapted from Figs. 4 and 5 of the original article) shows the different ER Ca2+ stores activated by MeCh and TSG stimulation. In Fig. 3A, 10 µM MeCh elicited a transient Ca2+ response in nominal Ca2+-free solution. After that, varying TSG concentrations (0.015, 0.03, and 1 µM) were added, and a second Ca2+ response was generated (traces 1–3; Fig. 3A). The presence of the second Ca2+ response indicates that TSG was able to induce additional ER Ca2+ release after the store sensitive to MeCh was depleted. Finally, the addition of 3 mM Ca2+ to the bathing solution resulted in a sustained Ca2+ plateau. It was shown previously in Fig. 2A that ER Ca2+ depletion was complete even if TSG concentration varied from 0.03 to 1 µM, and a similar amplitude of Ca2+ influx was seen upon the addition of extracellular Ca2+. The same is shown in Fig. 3A, where 0.015, 0.03, and 1 µM TSG all depleted the ER Ca2+ store to a similar extent, as evidenced by the superimposing Ca2+ plateau traces in the presence of 3 mM Ca2+ (traces 1- 3). When no TSG was added (trace 4; Fig. 3A), only the MeCh-sensitive ER Ca2+ store was depleted, and this was not enough to trigger maximal CCE, as seen in traces 1–3.


Figure 4
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  		Fig. 4. Dual effects of La3+ on MeCh- and TSG-elicited Ca2+ responses. A: MeCh-elicited Ca2+ responses in nominal Ca2+-free solution with varying La3+ concentrations added. Ca2+ (1 mM) was added sequentially to the bathing solution in trace 1 after the MeCh response. In traces 2-4, 3 mM Ca2+ was added extracellularly. B: comparison of MeCh-elicited Ca2+ responses in 1 mM Ca2+ with and without prior application of La3+ followed by the addition of 2 mM Ca2+ to the extracellular space. C: effects of La3+ on TSG-elicited Ca2+ responses in nominal Ca2+-free solution. In traces 1–3, La3+ was added at different points as indicated. See the teaching notes for the individual La3+ concentrations used and the corresponding traces.

 

Figure 5
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  		Fig. 5. Effect of La3+ on ER Ca2+ recycling. A: Ca2+ responses elicited by MeCh (traces 2 and 3) and TSG (traces 1–3) with the application of La3+ (points a–c). In traces 2 and 3, atropine (ATR) was added to terminate the MeCh response. B and C: successive MeCh-elicited Ca2+ responses (with washouts in between) in nominal Ca2+-free solution. La3+ was present in one of the graphs (B or C; see the teaching notes). See the teaching notes for the points of La3+ application and the corresponding traces.

 
Figure 3A shows that a portion of the Ca2+ store (from within the ER or other intracellular Ca2+ compartments) was not accessible to MeCh and was released only upon TSG application. That the TSG-sensitive ER Ca2+ store also includes the MeCh-sensitive one is demonstrated in Fig. 3B. The application of 0.1 µM TSG elicited a transient Ca2+ response that was not followed by a MeCh-induced response (trace 1; Fig. 3B), suggesting that most if not all of the ER Ca2+ store was already depleted by TSG stimulation. A lower TSG concentration (0.03 µM) also depleted most of the ER Ca2+ store (trace 2; Fig. 3B). The subsequent addition of MeCh only generated a very small response as a result (trace 2). At 0.015 µM, TSG induced partial depletion of the ER Ca2+ store only, leaving plenty of Ca2+ to be released by MeCh stimulation (trace 3; Fig. 3B). It is interesting to note that after the additions of both TSG and MeCh, the ER Ca2+ store was depleted to a similar extent, allowing a comparable amount of Ca2+ influx afterward with 3 mM extracellular Ca2+. The amplitudes of CCE, after ER Ca2+ store depletion stimulated by TSG and MeCh, were also comparable among traces 1–3 in Fig. 3A and traces 1–3 in Fig. 3B. In the traces mentioned, MeCh induced partial depletion of the ER Ca2+ store while the remaining ER Ca2+ was released by the addition of TSG. In the absence of TSG (trace 4; Fig. 3A), the ER still contained a significant amount of Ca2+ after MeCh stimulation, thus reducing the CCE response.

The distinction between MeCh-sensitive and MeCh-insensitive (i.e., portion of the TSG sensitive) ER Ca2+ stores is also demonstrated in Fig. 3, C and D. Subsequent to the activation of the muscarinic receptor by MeCh, inositol 1,4,5-trisphosphate (IP3) is synthesized and was used here to trigger ER Ca2+ release directly. Cells were permeabilized with saponin to facilitate the delivery of IP3 and heparin (an IP3 receptor antagonist), both of which are membrane impermeable. Figure 3C shows the partial ER Ca2+ release upon the addition of IP3 to mimick MeCh stimulation at the muscarinic receptor. The subsequent addition of TSG further induced ER Ca2+ release. However, no IP3-induced ER Ca2+ release was visible when TSG was added first to elicit a response (Fig. 3D).

Student handout 3. Figure 3 is included in the handout.

  1. Given 0.015, 0.03, and 1 µM of TSG were added in Fig. 3, A and B, match the TSG concentration with the correct trace.
  2. Why is the last Ca2+ response in trace 4 smaller than those in traces 1–3 in Fig. 3A?
  3. Why do the amplitudes of the MeCh-induced Ca2+ responses differ in Fig. 3B despite the same MeCh concentration being added?
  4. Both IP3 (used in Fig. 3, C and D) and MeCh (used in Fig. 3, A and B) elicited the same kind of responses with respect to TSG addition before or after treatment. In terms of Ca2+-mobilizing effects, how is IP3 related to MeCh?
  5. What can be concluded about the nature of the MeCh-sensitive ER Ca2+ store in relation to the TSG-sensitive one?

Dual Effects of La3+ in Blocking Ca2+ Extrusion and Ca2+ Influx

Teaching points for student handout 4. We have seen in the above sections that the extent of CCE, or Ca2+ influx after store depletion, depends on extracellular Ca2+ concentration. Another factor, cytosolic Ca2+ removal, also determines the amplitude and rate of CCE. Figure 4 (adapted from Figs. 6 and 7 of the original article) made use of La3+ to show the importance of both Ca2+ influx and cytosolic Ca2+ retention in generating Ca2+ responses.

Figure 4A shows that La3+ (from 0.03 to 0.5 mM) blocked Ca2+ influx in the presence of 3 mM extracellular Ca2+ after MeCh-induced ER Ca2+ release in nominal Ca2+-free solution (traces 2-4). Trace 1 in Fig. 4A shows the presence of CCE, which increased in amplitude as the extracellular Ca2+ concentration was raised, when La3+ was not added. In addition to blocking Ca2+ influx, La3+ was shown to retard the Ca2+ decay phase after MeCh stimulation. Cytosolic Ca2+ extrusion was prevented by La3+. More cytosolic Ca2+ was retained in the cytosol as the La3+ concentration was increased from 0.03 to 0.5 mM in traces 2-4 (Fig. 4A). More Ca2+ is taken up by the ER and is available for release, contributing to the elevated Ca2+ responses elicited by MeCh in traces 3 and 4 (compared with trace 2).

A typical biphasic Ca2+ response elicited by MeCh in 1 mM extracellular Ca2+ is shown in Fig. 4B in trace 1. The sustained plateau phase (immediately after the initial upstroke) indicates that Ca2+ influx after the ER Ca2+ store is partially depleted by MeCh. Increasing the extracellular Ca2+ concentration to 2 mM results in greater Ca2+ influx (trace 1), as shown earlier. In trace 2 in Fig. 4B, 0.5 mM La3+ was added before the MeCh response. As cytosolic Ca2+ removal was inhibited, a larger Ca2+ response is seen (trace 2). The higher peak Ca2+ and slower Ca2+ decay indicate greater Ca2+ availability in the cytosol that is taken up by the ER for release. The addition of 2 mM extracellular Ca2+ afterward failed to elicit a response (trace 2; Fig. 4B), in agreement with the responses shown in Fig. 4A.

Knowing the potential facilitatory effect of La3+ on ER Ca2+ uptake and release, it would be interested to examine whether cytosolic Ca2+ retention is affected by the extent of ER Ca2+ depletion. The rapid initial upstroke of the MeCh-induced Ca2+ response prevents the application of La3+ at different points of the ER Ca2+-depleting process. The time course of the TSG-induced Ca2+ response is much slower, and so the ER Ca2+-ATPase inhibitor was used here. Figure 4C shows traces of TSG-elicited Ca2+ responses in nominal Ca2+-free solution when 0.5 mM La3+ was added at different points of ER Ca2+ depletion. Trace 1 in Fig. 4C shows that when La3+ was added before TSG, the resultant Ca2+ response was much greater and had a very slow decay phase. If La3+ was added after the TSG-induced Ca2+ maximum had been reached, a secondary Ca2+ response was seen followed by a slow decay phase (trace 2; Fig. 4C). The addition of La3+ after the completion of the TSG response failed to elicit any response (trace 3; Fig. 4C). Altogether, blockade of Ca2+ extrusion is most effective before ER Ca2+ release, i.e., when [Ca2+]i is at the resting level. After exiting the ER, Ca2+ would be lost to the extracellular space, and the addition of La3+ would not retain the Ca2+ in the cytosol (trace 3; Fig. 4C). If Ca2+ extrusion is stopped prematurely by La3+, some Ca2+ retention would occur, and subsequent ER Ca2+ release would be enhanced (trace 2). By preventing any Ca2+ extrusion before stimulating ER Ca2+ release, the amount of Ca2+ mobilized to the cytosol would be maximal (trace 1).

Based on the above, in reference to Fig. 4C, the Ca2+ responses resulting from the addition of La3+ at other points can be predicted. Two sample scenarios are given. First, La3+ is added at point a (Fig. 4C), i.e., slightly after the initiation of the TSG response. The Ca2+ response would follow that of trace 2 up to point a, where it would deflect upward due to La3+-induced Ca2+ retention, followed by a slow decay phase. The maximal Ca2+ attained will be higher than that in trace 2 since less Ca2+ is lost to the extracellular space before La3+ is added. On the other hand, maximal Ca2+ will be lower than that of trace 1, where no Ca2+ extrusion ever took place. In the second scenario, La3+ is added at point b (Fig. 4C), i.e., at the peak of the TSG response. A secondary Ca2+ response may or may not be visible because significant ER Ca2+ release would have already occurred and was thus lost to the extracellular space. The addition of La3+ may only retain the remaining Ca2+ and retard the decay phase of the response.

Student handout 4. Figure 4 is included in the handout.

  1. What effects does La3+ have in regulating Ca2+ flux in a cell?
  2. In Fig. 4A, which traces were under the influence of La3+? Of these traces, which one was obtained with the highest and lowest La3+ concentration, respectively?
  3. Explain why traces 3 and 4 in Fig. 4A show a slower Ca2+ decay phase.
  4. Explain why trace 2 in Fig. 4B fails to show a response to the addition of 2 mM Ca2+.
  5. In Fig. 4C, why do the responses in traces 1–3 differ when La3+ was added at different points?
  6. With reference to Fig. 4C, predict the Ca2+ response if La3+ is added at points a and b.

Facilitation of ER Ca2+ recycling by La3+

Teaching points for student handout 5. The concept of blocking Ca2+ extrusion to retain Ca2+ for ER Ca2+ recycling was confirmed in the experiments shown in Fig. 5A (adapted from Figs. 8A, 9, and 10 of the original article). Figure 5A compares the effect of La3+ before and after MeCh application. Complete Ca2+ retention by La3+, added at point c before any ER Ca2+ release, was obtained in trace 3 in Fig. 5A. Maximal Ca2+ mobilization after TSG application was seen as a result. The addition of La3+ before MeCh (at point a) yielded a Ca2+ response that decayed slowly (trace 2) compared with trace 1, where La3+ was added only after the MeCh response (at point b; Fig. 5A). The subsequent TSG-induced Ca2+ response in trace 2 was also larger than in trace 1. The larger TSG response in trace 2 indicates Ca2+ was retained after MeCh-induced release. Trace 2 shows direct evidence of the retained Ca2+ being taken up by the ER for further release or, in other words, ER Ca2+ recycling. In trace 1, after MeCh stimulation, Ca2+ loss to the extracellular space was not prevented, resulting in a less filled ER Ca2+ store and thus a smaller TSG response.

Figure 5, B and C, can be discussed in parallel to highlight the facilitatory role of La3+ in ER Ca2+ recycling in the absence of extracellular Ca2+. After a transient Ca2+ response was elicited and after a washout period in nominal Ca2+-free solution, MeCh was unable to cause further Ca2+ release (Fig. 5B). However, in the presence of La3+, MeCh not only elicited successive Ca2+ responses, but the responses were sustained also (Fig. 5C). The responses in Fig. 5C again demonstrate that La3+ inhibits cytosolic Ca2+ extrusion, allowing ER Ca2+ recycling to occur for subsequent MeCh-induced release. If extracellular Ca2+ is added (arrows in Fig. 5), CCE is expected under the conditions shown in Fig. 5B but not Fig. 5C, because La3+ blocks Ca2+ influx in the latter.

Student handout 5. Figure 5 is included in the handout.

  1. In each of the three traces shown in Fig. 5A, La3+ was added at different points (points a–c). Match the point of La3+ addition with the correct trace. Explain how La3+ affects MeCh- and TSG-induced Ca2+ responses.
  2. In Fig. 5, B and C, which graph has La3+ added? Describe how La3+ affects the first and second, if any, MeCh-induced Ca2+ response.
  3. What can be concluded about the effect of La3+ on ER Ca2+ recycling?
  4. If extracellular Ca2+ is added at the end of the experimental trace (indicated by arrows) in both Fig. 5, B and C, what can be expected of the Ca2+ level?

SUMMARY

This teaching article outlines how the concept of intracellular Ca2+ movements, including ER Ca2+ release, ER Ca2+ recycling, and CCE, can be taught using figures adapted from Kwan et al. (8). This article is divided into five sections, each consisting of an explanation of the figures that serve as teaching notes followed by a student handout with learning questions. For those interested in the field of Ca2+ regulation pertaining to CCE, this article (as well as the original article) is particularly helpful in understanding the pathway of Ca2+ transport in refilling the ER after store depletion.

Received for publication April 7, 2009. Accepted for publication May 6, 2009.

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