A presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP exchange factor msec7-1

Abstract

ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane.

Figure 1

Plasma membrane localization of ARF6/msec7-1 complex. HEK 293 cells were transfected with expression vectors of full length ARF6 with C-terminally attached GFP (ARF6-GFP) (A) or a combination of ARF6-GFP and msec7-1 (B). Confocal fluorescence images are displayed on the left; transmitted light images of the same cells are displayed on the right. While ARF6-GFP alone was associated mainly with endosomes and partially along the plasma membrane (A), the ARF6-GFP/msec7-1 complex was located almost exclusively to the plasma membrane (B). (Bar = 10 μm.)

Figure 2

msec7-1 is located predominantly to presynaptic regions of Xenopus nerve cells. Shown is the fluorescence pattern of spinal cord neurons injected with a mixture of msec7-1 and GFP mRNA (A) or with msec7-1-GFP mRNA (B). Note that the postsynaptic muscle cells in these examples are not visible because of their lack of GFP fluorescence. Arrows, varicosities; arrowheads, cell bodies. (Bar = 50 μm.) (C) Quantitative analysis of fluorescence intensities. The ratio of varicosity to soma fluorescence (f_R) in GFP and msec7-1-GFP containing neurons was 0.87 ± 0.11 (n = 8) and 1.39 ± 0.24 (n = 8), respectively. Error bars are given as SEM.

Figure 3

msec7-1 expressing synapses have a higher frequency of spontaneous synaptic currents. (A) Representative traces of SSCs recorded from muscle cells that were innervated either by a control neuron (upper left) or by an msec7-1-containing neuron (lower left). The currents on the muscle cells are shown as downward deflections and represent single SSCs. (Bars = 0.5 s and 0.5 nA.) The corresponding amplitude distributions of the SSCs are shown on the right. (B and C) Summary of SSC frequency (B) and amplitude (C) of control and msec7-1-containing synapses. The average SSC frequency was 0.1 ± 0.02 Hz in control neurons (mean ± SEM; 153 SSCs, 13 cells) and 0.5 ± 0.12 Hz in msec7-1 neurons (mean ± SEM; 892 SSCs, 17 cells). The average SSC amplitude was 281 ± 22.7 pA and 345 ± 11.5 pA (mean ± SEM) for control and msec7-1 neurons, respectively. Error bars are given as SEM.

Figure 4

Initial EPSC amplitudes are increased in msec7-1-containing synapses. (A) Representative experiment of an msec7-1-containing synapse. Trains of six action potentials were elicited on the nerve cell soma, and the resulting EPSCs were recorded from the innervated muscle cell. Data points represent the average amplitude of the EPSCs obtained during each train. The apparent fast rundown in amplitude is also evident from raw traces of first EPSCs in the indicated trains (Inset). (Bars = 5 ms and 5 nA.) The lower panel illustrates the series resistance values recorded from the muscle cell during the experiment. (B) Averaged, absolute EPSC amplitudes of control (open circles; n = 27) and msec7-1-containing synapses (filled circles; n = 26). Each data point represents the mean value of six EPSC amplitudes obtained during each train averaged for all experiments. (C) Same data as in B normalized to the first amplitude. The rundown in msec7-1 neurons has time constants of 15.7 and 125 s whereas in control cells the rundown is much slower and could be fitted with one exponential with a time constant of 180 s. (D) Comparison of averaged EPSC amplitudes, normalized to the first amplitude, of control (open circles; n = 27) and msec7-1-mut-containing synapses (filled squares; n = 13). Notably, no statistically significant difference in rundown of EPSC amplitudes could be observed between control and msec7-1-mut cells. Error bars are given as SEM.