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E-Conference: Defining Calcium Entry Signals

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Physiological agonist-concentrations

18 June 2004

Trevor Shuttleworth

Indu: some comments...

1. "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."

I would disagree. As I have already noted on this forum, we have measured the Ca2+-selective currents responsible for Ca2+ entry at agonist concentrations that are just able to produce the very weakest Ca2+ signals (see JBC 276: 35676; and Biochem Soc Trans. 31: 916). These currents show all the unique specific characteristics of the ARC channels, and are entirely distinct from the currents activated by store-depletion (SOC channel currents).

2. "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."

Again, I would disagree. This is certainly true for pancreatic acinar cells. However, in parotid acinar cells, Dave Yule's group have shown that although the Ca2+ signals originate apically, they quickly spread throughout the cell in less than 200 ms (J. Physiol. 540:469).

3. "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+."

I think this is more of an apparent difference than a real one. As you note later in your posting, in almost all cases, the oscillations themselves result from the cyclical release and re-uptake of Ca2+ into the stores. Based on the evidence we, and others, have accumulated over recent years, the role of Ca2+ entry seems to be simply to facilitate the likelihood of this happening.

In other words, the main effect of Ca2+ entry under these conditions is to modulate the frequency of the oscillations. In some cases this influence is "absolute" in that oscillations cease immediately external Ca2+ is removed. In other cases oscillations continue, often for a considerable period, in the absence of external Ca2+. But even in these latter cases, the rate of Ca2+ entry modulates oscillation frequency.

Simply put, although the fundamental mechanism underlying the oscillations themselves may be independent of entry, their frequency is almost always very dependent on the rate of entry.

4. "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."

We have done precisely this, at least in HEK293 cells, and the results are published (see my response #1 above). As noted, the currents activated at low agonist concentrations are NOT the same as those activated at high concentrations. Moreover, their mode of activation is also entirely different.

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