Figure 2. Calcium signaling in Xenopus muscle cell and synaptic homeostasis.

The middle scheme depicts our model of homeostatic control of the synaptic strength. The nicotinic Ca²⁺ influx elicits a positive retrograde feedback signal (red arrow) on the presynaptic neurotransmitter release. The positive feedback is balanced by a negative retrograde feedback signal (blue arrow) triggered by the muscle AP-induced DICR. (DICR = depolarization-induced calcium release, AP = action potential, RyR = ryanodine receptor, SR = sarcoplasmic reticulum, R nico = nicotinic receptors). (A) Independence of the DICR signal from external Ca²⁺. AP (black trace, induced with a brief postsynaptic current step), Ca²⁺ dye (Fluo4) relative fluorescence in standard external medium (dark blue trace) and in Ca²⁺ free medium (light blue trace), and qualitative measure of the cell contraction (green trace, see Materials and methods). (B) Voltage-dependence of the DICR signal. The Fluo4 fluorescence was measured at the steady-state of the Ca²⁺ signal during a classical voltage-step protocol from a holding potential of -80 mV and averaged (n = 3 cells). (C) Blockade of the DICR signal by loading the muscle cell with 100 µM ryanodine. (D) Representative example of the DICR signal (blue trace) during repetitive muscle APs (black trace). (E) Ca²⁺ build-up upon nicotinic receptor activation. Fluo4 signal (upper traces) in control and in Ca²⁺ free medium, during iontophoretic ACh applications (5 pulses) under postsynaptic voltage-clamp (-80 mV). The lower trace shows the nicotinic currents. (F) Dependence of the nicotinic Ca²⁺ build-up on the nicotinic conductance with increasing iontophoretic ACh applications at a constant membrane potential (holding potential –80 mV).

DOI: http://dx.doi.org/10.7554/eLife.12190.007

Figure 2—figure supplement 1. Ionic permeability of the Xenopus nicotinic receptor-channel.

To determine the ionic selectivity of the Xenopus nicotinic receptor in cultured cells, the reversal potential of the nicotinic current induced by ionophoretic acetylcholine application was measured under voltage-clamp in solutions of various ionic compositions, with an intra-pipette medium of the following composition (in mM): 110 KCl, 3 NaCl, 2 MgCl2, 0.5 EGTA and 10 HEPES (pH 7.2). (A) Current-voltage relationship of the nicotinic current induced by ionophoretic application of acetylcholine, in a classical physiological medium. Inset shows the current traces recorded at the different holding potentials. (B) Table summarizing the external media compositions and the corresponding reversal potentials (Vr). Concentrations are expressed in mM, and the mean ± SEM reversal potentials in mV. Fitting the measured reversal potentials to the GHK voltage equation (see Materials and methods) gave: PK/PNa = 1.07, PCa/PNa = 0.23, and PMg/PNa = 0.31, used to calculate the theoretical reversal potentials t-Vr.

DOI: http://dx.doi.org/10.7554/eLife.12190.008