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E-Conference: Defining Calcium Entry Signals

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Comments on cellular domains that contribute to calcium entry events

15 June 2004

Jim Putney

I am commenting in response to Mike’s contribution, but really I have something general to say that is relevant to several of the contributions. I wish to speak to two issues that have been mentioned on several occasions. These are (1) it is best to address signaling mechanisms for physiological concentrations of hormones and neurotransmitters, and (2) it is always better to measure current directly than to deal with fluxes based on fluorescent indicators.

Of course, no one can be opposed to studying physiological concentrations of agonists; this would be akin to being opposed to motherhood. Nonetheless, at lower agonists concentrations some difficulties arise.

For the case of the store-operated channels, I really do not know of a single instance in which anyone has measured the current underlying these channels under truly physiological conditions. In many cell types, even the extreme manipulations of high intracellular Ca2+ buffering do not bring up enough current to be reliably measured. Yet the fluorescence experiments indicate it is clearly there.

The reason for this is that with all of the potentials problems and artifacts from the use of fluorescent indicators (all of which can be dealt with by use of appropriate controls), this is still by far a much more sensitive measurement, for Ca2+ anyway, than measurement of current. Thus as the agonist concentrations get lower and lower, the experiments and evidence for and against various proposed mechanisms will of necessity get less direct.

Under these conditions, it is useful to employ specific pharmacological tools to identify specific pathways. I am not sure that I agree with the general statement that we do not have a specific blocker of SOCs.

It is true we do not have a single, highly specific and potent inhibitor, such as the dihydropyridines or TTX. However, I would suggest that a complete block by both very low (1 um and below) concentrations of lanthanides together with block by 2APB (2-aminoethoxydiphenyl borate) in the 20-50 uM range is diagnostic for store-operated channels. I do not know of any other channel with these pharmacological properties (but perhaps one of the other participants does).

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