• You have just completed making several figures illustrating a series of your experiments.

  • using the ratio-metric $C{a}^{+2}$$Ca^{+2}$-sensitive fluorophore Fura-2

• measure changes in cytosolic $C{a}^{+2}$$Ca^{+2}$ during receptor-mediated activation of lacrimal gland cells

• But unfortunately you have misplaced the information concerning when chemicals were added to the bathing solutions

• But you do remember that methacholine is a muscarinic cholinergic receptor agonist

• You plan to enlist the help of your N&P Study Group to refresh your memory by using their combined physio-logic.

• The List of Chemicals Used Was :

  • methacholine ( MeCh )

    • muscarinic receptor agonist

    • stimulates Gq-coupled receptors , leading to IP3-mediated calcium release from ER

  • atropine ( Atr )

    • muscarinic receptor antagonist

    • blocks MeCh's effect by preventing receptor activation

  • inositol triphosphate ( IP3 )

    • second messenger that binds to IP₃ receptors on the ER ,

      • causing Ca²⁺ release into the cytoplasm

  • $C{a}^{+2}$$Ca^{+2}$

  • $L{a}^{+3}$$La^{+3}$

    • Ca²⁺ channel blocker

    • used to inhibit store-operated Ca²⁺ entry ( SOCE ) across the plasma membrane

  • thapsigargin ( TSG )

    • SERCA pump inhibitor

    • which blocks ER Ca²⁺ reuptake ,

      • causing ER Ca²⁺ depletion and activation of SOCE

  • EGTA

    • A Ca²⁺ chelator

    • high selectivity for Ca²⁺ over Mg²⁺

    • used to buffer extracellular Ca²⁺ and limit its effects

  • ionomysin ( Iono )

    • calcium ionophore that facilitates Ca²⁺ transport across membranes ,

      • raising intracellular Ca²⁺ levels independently of receptors

  • heparin

    • used intracellularly as an IP₃ receptor antagonist ,

      • blocking Ca²⁺ release from the ER by preventing IP₃ binding

• Each trace represents the time course of the $C{a}^{+2}$$Ca^{+2}$ response from an experiment with a batch of lacrimal gland cells

  • A: MeCh-elicited $C{a}^{+2}$$Ca^{+2}$ responses with and without extracellular $C{a}^{+2}$$Ca^{+2}$

  • B: Addition of a drug at the arrows indicated abolished the MeCh-elicited $C{a}^{+2}$$Ca^{+2}$ response.

1. In Figure 1A , the additions at time points $a$$a$ through $d$$d$ were either extracellular $C{a}^{+2}$$Ca^{+2}$ or EGTA for traces 1 through 3. Time $c$$c$ was an addition to trace 2 , and time $d$$d$ was an addition to trace 3. Provide the best choices for each time point.

   • Initial Spike = intracellular calcium

   • Plateau = extracellular calcium

   

Extracellular $C{a}^{+2}$$Ca^{+2}$ was present during only some of the traces in Figure 1A. ( ok )

2. In Figure 1B , the same chemical was added to each trace ( at the points indicated by arrows ). Was extracellular $C{a}^{+2}$$Ca^{+2}$ present ?

   • yes , extracellular was present

   • there is still a sustained plateau after the initial spike

   

• A: TSG-induced $C{a}^{+2}$$Ca^{+2}$ responses in nominally $C{a}^{+2}$$Ca^{+2}$ -free solution.

  • The range of TSG concentrations used was from 0.0 µM to 1.0 µM , which correspond to traces 1 through 6

• B: TSG responses with varying extracellular $C{a}^{+2}$$Ca^{+2}$ concentrations

  • The $C{a}^{+2}$$Ca^{+2}$ concentrations used were :

    • 0.3 mM in trace 3

    • 1.0 mM in trace 4

    • 3.0 mM in trace 5

• Extracellular $C{a}^{+2}$$Ca^{+2}$ was not present for traces 1 and 2.

• EGTA was present in trace 1 only.

3. In Figure 2 the additions at points a , b , and c were either $C{a}^{+2}$$Ca^{+2}$ or EGTA. Provide the best choices for each time point

   • Time-Point-A = calcium added

   • Time-Point-B = EGTA added

   • Time-Point-C = extracellular calcium added

4. In Figure 2A , a specific TSG concentration ( 0.0 ; 0.01 ; 0.03 ; 0.3 ; 0.7 ; or 1.0 µM ) was added for each trace , 1 through 6. Match each concentration to the trace number.

   • blocking calcium export , results in high levels of intracellular calcium

   • Trace 1 = 1.0 µM

   • Trace 2 = 0.7 µM

   • Trace 3 = 0.3 µM

   • Trace 4 = 0.03 µM

   • Trace 5 = 0.01 µM

   • Trace 6 = 0.0 µM

     • calcium wasn't depleted , so ORI never reached out to STIM

       • it only connects if calcium is low in the ER

5. In Figure 2A , the TSG-induced $C{a}^{+2}$$Ca^{+2}$ response of trace 4 must have been smaller and slower than those in traces 1 through 3 for a reason. Convince your group what was going on. What evidence supports whether complete ER $C{a}^{+2}$$Ca^{+2}$ depletion occurred ?

   • TSG is a SERCA pump inhibitor

     • so calcium is never removed from cytosol and stored in ER

   • Trace 4 we thought had lower amount of TSG ( 0.03 µM ) ,

     • so its not going to inhibit SERCA pump

     • therefore , there should be very little intracellular calcium

   • So even though it has slower kinetics , when we added calcium back in at Time-Point-A , we see a significant calcium level increase ( SOCE )

6. In Figure 2B , predict the level of $C{a}^{+2}$$Ca^{+2}$ influx for traces 1 , 3 , and 4. Also predict if the extracellular $C{a}^{+2}$$Ca^{+2}$ concentration had been increased to 3 mM at time point c.

   • Trace 1 and 2 = no influx

   • Trace 3 = small influx

   • Trace 4 = moderate influx

   • If you added extra extracellular calcium at Time-Point-C ,

     • then trace 2 would take a very long time to return to baseline

     • trace 6 would also plateau more , instead of declining

• A: Following MeCh-induced $C{a}^{+2}$$Ca^{+2}$ responses , varying TSG concentrations were added in nominally $C{a}^{+2}$$Ca^{+2}$ -free solution.

  • Extracellular $C{a}^{+2}$$Ca^{+2}$ was added to elicit the final $C{a}^{+2}$$Ca^{+2}$ response.

• B: $C{a}^{+2}$$Ca^{+2}$ responses induced by varying TSG concentrations were followed by MeCh application.

  • Extracellular $C{a}^{+2}$$Ca^{+2}$ was added to elicit the final $C{a}^{+2}$$Ca^{+2}$ response.

7. In Figure 3A-B , match the TSG concentration ( 0.0 , 0.015 , 0.03 , 1.0 µM ) with traces 1 through 4

   • Panel A :

     • Trace 1 = 1.0 µM

     • Trace 2 = 0.03 µM

     • Trace 3 = 0.015 µM

     • Trace 4 = 0.0 µM

   • Panel B :

     • Trace 1 = 1.0 µM

     • Trace 2 = 0.03 µM

     • Trace 3 = 0.015 µM

8. In Figure 3A , what caused the response to extracellular $C{a}^{+2}$$Ca^{+2}$ in trace 4 to be smaller than these last responses seen in traces 1 through 3 ?

   • Trace 4 had zero TSG

     • so ER calcium stores were never depleted

       • therefore STIM / ORAI were never activated

   • so even after adding extracellular calcium , there is little flux

9. In Figure 3B , what caused the amplitudes of the MeCh-induced $C{a}^{+2}$$Ca^{+2}$ responses to differ despite the same MeCh concentration being added ?

   • High amounts of TSG ➡️ fully deplete ER calcium ➡️ no calcium left for MeCH to release

   • Low amounts of TSG ➡️ ER calcium available ➡️ MeCH response

• A: MeCh-elicited $C{a}^{+2}$$Ca^{+2}$ responses in nominally $C{a}^{+2}$$Ca^{+2}$-free solution with varying $L{a}^{+3}$$La^{+3}$ concentrations added

  • $C{a}^{+2}$$Ca^{+2}$ ( 1 mM ) was added sequentially to the bathing solution in trace 1 after MeCh

  • In traces 2 through 4 , extracellular $C{a}^{+2}$$Ca^{+2}$ was added at 3 mM

• B: Comparison of MeCh-elicited responses in 1 mM extracellular $C{a}^{+2}$$Ca^{+2}$ , with or without prior application of $L{a}^{+3}$$La^{+3}$

  • followed by addition of $2mMC{a}^{+2}$$2\ mM\ Ca^{+2}$ to the extracellular solution

• C: Action of $L{a}^{+3}$$La^{+3}$ on TSG-induced responses in nominally $C{a}^{+2}$$Ca^{+2}$-free solution

  • $L{a}^{+3}$$La^{+3}$ was added at different time points during traces 1 through 3

10. How does $L{a}^{+3}$$La^{+3}$ influence $C{a}^{+2}$$Ca^{+2}$ flux into and out of a cell ?

    • $L{a}^{+3}$$La^{+3}$ :

      • blocks calcium influx by competing with calcium entry

      • slows calcium efflux by inhibiting PMCAs

11. In Figure 4A , in which traces was $L{a}^{+3}$$La^{+3}$ present? Rank order these traces from the lowest to highest $L{a}^{+3}$$La^{+3}$ concentration ( 0.0 , 0.03 , 0.2 , 0.5 µM )

    • Trace 1 = 0.0 µM || Zero $[L{a}^{+3}]$$[\ La^{+3}\ ]$

    • Trace 2 = 0.03 µM || Lowest $[L{a}^{+3}]$$[\ La^{+3}\ ]$

    • Trace 3 = 0.2 µM || Intermediate $[L{a}^{+3}]$$[\ La^{+3}\ ]$

    • Trace 4 = 0.5 µM || Highest $[L{a}^{+3}]$$[\ La^{+3}\ ]$

12. In Figure 4A , what caused traces 3 and 4 to have slower $C{a}^{+2}$$Ca^{+2}$ decay phases than trace 2 ?

    • the high amount of $[L{a}^{+3}]$$[\ La^{+3}\ ]$ inhibits PMCAs

      • reducing calcium export

        • this exaggerates the plateau on the trace

13. In Figure 4B , what caused the observed response in trace 2 to addition of 2 mM $C{a}^{+2}$$Ca^{+2}$ ?

    • $L{a}^{+3}$$La^{+3}$ is still around and is blocking STIM / ORAI channels

14. In Figure 4C , what led to the difference in the responses for traces 1 through 3 when $L{a}^{+3}$$La^{+3}$ was added at different points along the time course ?

    • the earlier they add $L{a}^{+3}$$La^{+3}$ , the more effective / time it has to block internal calcium release

    • when they add it at the later times , calcium has more of a chance to exit internal storage locations

15. In Figure 4C , predict the $C{a}^{+2}$$Ca^{+2}$ response if $L{a}^{+3}$$La^{+3}$ is added at points a and b

    • Time-Point-A :

      • $L{a}^{+3}$$La^{+3}$ blocks SOCE , and it looks like Trace 1

    • Time-Point-B :

      • SOCE is partially blocked still , so it looks more like Trace 3