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E-Conference: Defining Calcium Entry Signals

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Physiological agonist-concentrations

17 June 2004

Indu S. Ambudkar

I think the discussion we have been having regarding concentrations of agonists used for Ca2+ influx measurements is a critical issue.

I agree with Reinhold and Jim (although, I would like to discuss the analogy to motherhood in greater detail !!!) that there are problems associated with our ability to accurately measure Ca2+ influx at low agonist concentrations. With the exception of one paper and different [IP3] in the pipette (Parekh) no-one has really reported the characteristics of channel activity at the so-called “physiological concentrations” of agonists.

What we do know from fura-2 measurements and readouts using K- or Cl channel activity is that typically these low concentrations induce oscillations, while higher concentrations give sustained [Ca2+]i elevations.

In polarized epithelial cells there is another level of complexity since the oscillations are spatially restricted. This might be due to the large abundance of IP3R in the luminal region or, as has been suggested, a more “sensitive” population of IP3Rs.

More importantly, and pertinent to this discussion, is the fact that the requirement for external Ca2+ in these oscillations appears to be different in different cells. In some cells, Ca2+ entry is absolutely required for the oscillations whereas in some cases the oscillations are relatively independent of external Ca2+. In the former, it is not entry that is oscillating per se, but rather each oscillation is due to the cycle of Ca2+ release via IP3R - activation of influx (meaning there must be inhibition of release, likely due to feed-back regulation of IP3R or PLC) - refill of stores - next release.

This in my opinion is the most interesting system. Ca2+ influx occurs without “global” Ca2+ changes even in the continued presence of the agonist. So, here is a store that can be depleted at low stimulus levels and is coupled to influx (see Reinhold’s point about IP3 sensitivity of the “Ca-store”).

The critical question of course is whether this Ca2+ entry is “store- operated” or activated by local changes in DAG/other messenger. This can be answered only by measurements of currents at these low concentrations to see if they are the same as those produced at higher agonist-concentrations. This is an important experiment that needs to be done to move forward in this field.

I also wanted to throw out one more point for discussion. What is really known about “physiological” concentrations of agonists ?

In fact, the junctional architecture of the region where neurotransmitters are released near the plasma membrane of cells can in fact enable fairly high local concentrations of agonists. If receptors are strategically localized in this region, depending on the affinity and number of such receptors, one could elicit a “High” but extremely localized response. Any thoughts……?

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