The electrical gradient across the mitochondrial inner membrane (⌿ m) is established by electron transport chain (ETC) activity and permits mitochondrial Ca 2؉ sequestration. Using rhodamine-123, we determined how repetitive nerve stimulation (100 Hz) affects ⌿ m in motor terminals innervating mouse levator auris muscles. Stimulation-induced ⌿ m depolarizations in wild-type (WT) terminals were small (<5 mV at 30°C) and reversible. These depolarizations depended on Ca 2؉ influx into motor terminals, as they were inhibited when P/Q-type Ca 2ϩ channels were blocked with -agatoxin. Stimulation-induced ⌿m depolarization and elevation of cytosolic [Ca 2؉ ] both increased when complex I of the ETC was partially inhibited by low concentrations of rotenone (25-50 nmol/l). This finding is consistent with the hypothesis that acceleration of ETC proton extrusion normally limits the magnitude of ⌿ m depolarization during mitochondrial Ca 2؉ uptake, thereby permitting continued Ca 2؉ uptake. Compared with WT, stimulation-induced increases in rhodamine-123 fluorescence were Ϸ5 times larger in motor terminals from presymptomatic mice expressing mutations of human superoxide dismutase I (SOD1) that cause familial amyotrophic lateral sclerosis (SOD1-G85R, which lacks dismutase activity; SOD1-G93A, which retains dismutase activity). ⌿ m depolarizations were not significantly altered by expression of WT human SOD1 or knockout of SOD1 or by inhibiting opening of the mitochondrial permeability transition pore with cyclosporin A. We suggest that an early functional consequence of the association of SOD1-G85R or SOD1-G93A with motoneuronal mitochondria is reduced capacity of the ETC to limit Ca 2ϩ -induced ⌿m depolarization, and that this impairment contributes to disease progression in mutant SOD1 motor terminals.amyotrophic lateral sclerosis ͉ calcium ͉ mitochondria ͉ motor nerve terminals M itochondria sequester significant amounts of stimulationinduced Ca 2ϩ loads in many cell types (1-9). This mitochondrial Ca 2ϩ uptake occurs via a Ca 2ϩ uniporter/channel (10) down a potential gradient (⌿ m , 150-200 mV, matrix negative) established by ETC activity (11,12). Entry of Ca 2ϩ into mitochondria depolarizes ⌿ m , which would be expected to reduce the gradient driving Ca 2ϩ uptake. However, in motor nerve terminals, mitochondrial Ca 2ϩ uptake continues throughout prolonged trains of action potentials (13). One possible explanation for this apparent paradox is that the ⌿ m depolarization produced by Ca 2ϩ entry reduces the gradient against which ETC complexes I, III and IV extrude protons, thus accelerating ETC proton extrusion (14). This acceleration would limit the net ⌿ m depolarization, thereby allowing mitochondria to continue taking up Ca 2ϩ even during prolonged stimulation (15). We tested this hypothesis in mouse motor terminals, and found that the ⌿ m depolarizations produced by repetitive stimulation at 50-100 Hz were Ca 2ϩ dependent and reversible, and were small (or undetectable) at near-physiological temperatures (30°C). Par...

Mitochondria sequester much of the Ca2+ that enters motor nerve terminals during repetitive stimulation at frequencies exceeding 10-20 Hz. We studied the post-stimulation extrusion of

Mitochondria in motor nerve terminals temporarily sequester large Ca2+ loads during repetitive stimulation. In wild-type mice this Ca2+ uptake produces a small (<5 mV), transient depolarization of the mitochondrial membrane potential (Ψm, motor nerve stimulated with at 100 Hz for 5 s). We demonstrate that this stimulation-induced Ψm depolarization attains much higher amplitudes in motor terminals of symptomatic mice expressing the G93A or G85R mutation of human superoxide dismutase 1 (SOD1), models of familial amyotrophic lateral sclerosis (fALS). These large Ψm depolarizations decayed slowly and incremented with successive stimulus trains. Additional Ψm depolarizations occurred that were not synchronized with stimulation. These large Ψm depolarizations were reduced (a) by cyclosporin A (CsA, 1-2 uM), which inhibits opening of the mitochondrial permeability transition pore (mPTP), or (b) by replacing bath Ca2+ with Sr2+, which enters motor terminals and mitochondria but does not support mPTP opening. These results are consistent with the hypothesis that the large Ψm depolarizations evoked by repetitive stimulation in motor terminals of symptomatic fALS mice result from mitochondrial dysfunction that increases the likelihood of transient mPTP opening during Ca2+ influx. Such mPTP openings, a sign of mitochondrial stress, would disrupt motor terminal handling of Ca2+ loads and might thereby contribute to motor terminal degeneration in fALS mice. Ψm depolarizations resembling those in symptomatic fALS mice could be elicited in wild-type mice following 0.5-1 hr exposure to diamide (200 μM), which produces an oxidative stress, but these depolarizations were not reduced by CsA.