Facilitation as a tool to study the entry of calcium and the mechanism of neurotransmitter release

We have shown the usefulness of using facilitation as an indirect tool for measuring release-related processes that cannot be measured directly. Most of the findings obtained by measuring facilitation have been verified (by various groups) by direct measurements in systems where such measurements could be carried out. This provides reassurance that the methodology is sound. Using facilitation one can gain insight into numerous processes that together govern the dependence of release on the intracellular calcium concentration C. The physiological conclusions have been listed in the text. We reiterate some of these conclusions here, in order to emphasize certain additional matters. For 20 years it has been known that release is a saturating cooperative function of the extracellular Ca²⁺ concentration C_e (Dodge and Rahamimoff, 1967). Yet several questions remained open, such as whether this behavior in fact reflected the dependence of the release on the intracellula Ca²⁺ concentration C, of entry on C_e, or of a combination of these individual possibilities. We have found, using short-term facilitation as the investigatory tool, that release is a saturating cooperative function of C. It is obvious that saturation will eventually take place as C increases. What is important to emphasize is that the saturation occurs at physiological values of C. Such values typically correspond to the amount of Ca²⁺ that enters following only one or very few pulses. Various aspects of facilitation F can be used to characterize the processes responsible for the removal of the calcium that enters the nerve terminal. Here we emphasized the role of the duration of F, but information can also be obtained by examining other aspects of facilitation (Parnas and Segel, 1980; H. Parnas et al. 1982; I. Parnas et al., 1982a). The principal conclusion is that removal shows saturation kinetics. This finding is strong evidence that diffusion is not the principal cause of removal, but rather directs attention to pumps and/or Na⁺ ⇄ Ca²⁺ exchange. It is rather surprising and very useful that facilitation can also help delineate the properties of Ca²⁺ entry. We have shown that measurements of the duration of facilitation can be very revealing, particularly when saturation in removal occurs following one or a few pulses. In addition, we have pointed out the advantages of a modified definition of facilitation in which the the first pulse varies in amplitude and the second pulse is fixed. Using these tools it was concluded that Ca currents saturate as a function C_e and show a bell-shaped dependence on membrane depolarization. The phenomenon of facilitation is not restricted to neurotransmitter release. Measurements of facilitation are possible in most secreting systems and as such provide an important alternative tool to direct measurements that are as yet largely impossible to perform.