Fluorescent calcium imaging or electrophysiology, or both?

4 June 2004

Randen L. Patterson

As a researcher who uses fluorescence and fura to study calcium signaling, I feel it necessary to retort a few of Trevor's comments.

First, these statements,
"The use of changes in cytosolic Ca2+ based on fluorescence measurements is simply not suitable to define a channel. Similarly, all the pharmacological tools currently available for such channels are so poor that they cannot be used with any confidence to discriminate between different pathways for entry. The only way we can even begin to characterize the pathways operating under various conditions in any given cell type is by measuring the channel properties themselves using electrophysiological techniques."
is not correct.

Electrophysiology (particularly whole-cell patching) can only provide the biophysical properties of the channel, not the molecular identity, unless coupled with other techniques, just as fluorescent calcium entry research. In addition, even if single channel recordings could be taken of these channels, it is performed in the ABSENCE of every other channel. Calcium signals are integrated into the receptor response, and have inputs and outputs to and from other ion channels. Therefore, study of single channels, although important to understanding channel properties, is a poor system for determining the "behavior" of these channels, or their role in cellular physiology.

In my opinion, fluorescent calcium imaging and electrophysiology are extremely complementary, and perhaps more "fura-people" should be teaming up with more "patchers", each lab using their expertise to truly begin getting a grip on what these channels do in physiology.

Secondly, what is the definition of calcium selective channels anyways?
Many of the researchers that study calcium entry channels can see ions, such as strontium and barium, passing into cells after receptor stimulation AT MILLIMOLAR CONCENTRATIONS!!! Strontium is found in human blood below 1 micromolar, and barium at 41-96 micromolar, while calcium is millimolar. Therefore, even if these channels are "non-selective" for the purposes of defining biophysical properties, in the body they are fluxing calcium, and should not be considered "non-selective" when trying to determine their role in cellular function.

I do agree with Trevor's comments on heteromultimer TRPC channels. As a field, this argument is bandied about all the time, although very few studies have addressed this exact question, not that it's an easy question to address. Only by trying to isolate homomultimer and heteromultimer TRPC channels in vivo, will we truly know the selectivity of these channels. Even with the advent of siRNA, it's still difficult to look at the loss of a current, as opposed to gaining a current.

Unfortunately, overexpression studies of TRPC channels are in part why we are in this quandary today. Each lab uses their own protocol for transient expression, all of which will differ in their expression as homomultimers and heteromultimers, and therefore provide different results. There is no easy answer to this problem, but that does not negate its importance.