THE INFLUENCE OF THE Na+/Ca2+ EXCHANGER DURING SHORT-TERM FACILITATION IN MOTOR NERVE TERMINALS

M.S.Desai1*; G.M.Sparks1,2; R.L.Cooper1

1. Dept Biol, Univ. of Kentucky, Lexington, KY, USA
2. Col. of Medicine, Univ. of Michigan, Ann Arbor, MI, USA

During chemical synaptic transmission residual [Ca2+]i due to previously evoked activity can accumulate in the nerve terminal. This is the basis of short-term facilitation (STF). Regulation of this [Ca2+]i is crucial to maintain normal vesicle fusion and transmitter release. Of the various Ca2+clearing mechanisms the plasmalemmal Na+/Ca2+ exchanger [NCX] and the Ca2+ATPase have been previously shown to have significant roles in controlling this residual calcium and thereby affect STF. We used the crayfish opener neuromuscular junction to address the role of the NCX in the development and maintenance of STF. We have demonstrated that there is a steady state in the amount of vesicles released after the induction of STF and the EPSPs rise and ultimately reach a plateau level. Moreover we have found that this release can be enhanced by reducing the rate of the NCX. The results support the idea that release is not saturated due to the lack in the ability of the terminal to release more transmitter or that there is a limitation in synaptic area for vesicle docking. Even though the electrochemical driving gradient for the EPSPs is still sufficient to result in larger potentials they are restricted because of the amount of transmitter being released. In order to index facilitation, the ratio of the peak amplitude of the EPSP for one of the preceding pulses from the last one within the stimulus train is used and a unitary value of 1 is subtracted from the ratio to provide the facilitation index (FI). When the NCX was compromised, the percent increase at 40Hz was 54% (A30/A5) and at 60Hz it was 34% (A30/A5). These results indicate that STF does in fact occur and that the NCX is crucial in shaping the extent of STF. The [Ca2+]i increases during the stimulation train but it is presumed that an equilibrium is reached with the continual influx, buffering and exchange mechanisms to result in a steady state of transmitter release. With a transition to a higher stimulation frequency it is likely a new equilibrium is established at a higher [Ca2+]i. The higher stimulation rate also results in a faster rise in [Ca2+]i, thus resulting in an increase in the mean quantal content and a faster rise of the EPSPs to the plateau level. We are interested in finding out the role of NCX in STF and in establishment of this equilibrium. We are currently examining the plateau phase of STF and its regulation by the NCX at various stimulation frequencies.

Support Contributed By: Dept of Biol. G. Ribble fellowship (MSD, GMS), Arnold and Mabel Beckman Foundation (GMS), and NSF-IBN-0131459 (RLC).

ABSTRACT 3

UNDERSTANDING CALCIUM REGULATION IN DROSOPHILA LARVAL HEART

Papoy A., Desai M. and Cooper R. L. Department of Biology, University of Kentucky

Calcium plays an important role in excitable cells. The concentration of calcium is particularly important in the heart where the ion (Ca2+) is necessary for maintaining periodic and synchronized contractions, thereby regulating the rythmicity of the heartbeat. In the cardiac myocytes, the calcium that enters has to be regulated as not to disrupt the functioning of these cells with an abnormally high calcium concentration. This calcium is removed from the cytosol either by driving it out of the cytosol, which is done by the means of various ion channels like the plasma membrane Ca2+ ATPase (PMCA) and the sodium calcium exchanger (NCX), or it can be sequestered into the sarcoplasmic reticulum via the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). I am interested in studying the differential contributions of the PMCA, NCX, and the SERCA in regulation of Drosophila larval heart rate.
In order to make accurate comparisons amongst the methods of calcium regulation, it is imperative to block (functionally compromise) each of the channels individually. A mutant fly line, Kum170 has a conditional mutation in the Drosophila SERCA gene, which when heat shocked causes the mutant SERCA flies fall into paralysis. This allows for a comparions between functioning and non functioning SERCA. In order to decrease the PMCA function the larvae are first dissected in normal fly HL3 saline at pH 7.2. Thereafter the saline is switched to one with pH 8.8 to block the PMCA. The heart rates are measured in both the salines allowing for comparison. Thereafter, in order to study the functional involvement of the NCX in regulation of the Drosophila larval heart rate, the concentration of Na+ in the saline will be reduced. The reduction of extracellular Na+ will cause a decrease in the driving gradient of the NCX and thus should also alter the heart rate.

PAPERS

Desai, M., Sparks, G., Dudel, J. and Cooper, R.L. (2007) The influence of the Na/Ca exchanger during short-term facilitation in motor nerve terminals (in preparation).