Work in heterologous systems has revealed that members of the Rad, Rem, Rem2, Gem/Kir (RGK) family of small GTP-binding proteins profoundly inhibit L-type Ca(2+) channels via three mechanisms: 1), reduction of membrane expression; 2), immobilization of the voltage-sensors; and 3), reduction of Po without impaired voltage-sensor movement. However, the question of which mode is the critical one for inhibition of L-type channels in their native environments persists. To address this conundrum in skeletal muscle, we overexpressed Rad and Rem in flexor digitorum brevis (FDB) fibers via in vivo electroporation and examined the abilities of these two RGK isoforms to modulate the L-type Ca(2+) channel (CaV1.1). We found that Rad and Rem both potently inhibit L-type current in FDB fibers. However, intramembrane charge movement was only reduced in fibers transfected with Rad; charge movement for Rem-expressing fibers was virtually identical to charge movement observed in naïve fibers. This result indicated that Rem supports inhibition solely through a mechanism that allows for translocation of CaV1.1's voltage-sensors, whereas Rad utilizes at least one mode that limits voltage-sensor movement. Because Rad and Rem differ significantly only in their amino-termini, we constructed Rad-Rem chimeras to probe the structural basis for the distinct specificities of Rad- and Rem-mediated inhibition. Using this approach, a chimera composed of the amino-terminus of Rem and the core/carboxyl-terminus of Rad inhibited L-type current without reducing charge movement. Conversely, a chimera having the amino-terminus of Rad fused to the core/carboxyl-terminus of Rem inhibited L-type current with a concurrent reduction in charge movement. Thus, we have identified the amino-termini of Rad and Rem as the structural elements dictating the specific modes of inhibition of CaV1.1.

BackgroundAmyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3–5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca2+ channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation.MethodsWe recorded L-type currents, charge movements, and myoplasmic Ca2+ transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1G93A).ResultsIn FDB fibers obtained from “symptomatic” global SOD1G93A mice, we observed a substantial reduction of SR Ca2+ release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1G93A fibers when compared to control fibers. Ca2+ transients were not significantly reduced in similar experiments performed with FDB fibers obtained from “early-symptomatic” SOD1G93A mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca2+ release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1G93A expression was restricted to skeletal muscle.ConclusionsWe report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1G93A mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1G93A and skeletal muscle autonomous MLC/SOD1G93A mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0094-6) contains supplementary material, which is available to authorized users.

Gaffield MA, Romberg CF, Betz WJ. Live imaging of bulk endocytosis in frog motor nerve terminals using FM dyes. J Neurophysiol 106: 599 -607, 2011. First published May 4, 2011 doi:10.1152/jn.00123.2011.-We observed endocytosis in real time in stimulated frog motor nerve terminals by imaging the growth of large membrane infoldings labeled with a low concentration of FM dye. The spatial and temporal information made available by these experiments allowed us to image several new aspects of this synaptic vesicle recycling pathway. Membrane infoldings appeared near synaptic vesicle clusters and grew rapidly during long-duration, highfrequency stimulation. In some cases, we observed large, elongated infoldings growing laterally into the terminal. We used these observations to calculate infolding growth rates. A decrease in stimulation frequency caused a decrease in growth rates, but the overall length of these structures was unaffected by frequency changes. Attempts to wash the dye from these infoldings after stimulation were unsuccessful, demonstrating that the fluorescent structures had been endocytosed. We also used this technique to trigger and image infoldings during repeated, short trains. We found that membrane uptake occurred repeatedly at individual endocytosis sites, but only during a portion of the total number of trains delivered to the terminal. Finally, we showed that phosphatidylinositol 3-kinase, but not actin, was involved in this endocytosis pathway. The ability to monitor many individual bulk endocytosis sites in real time should allow for new types of endocytosis measurements and could reveal novel and unexpected mechanisms for coordinating membrane recovery during synaptic activity. synaptic vesicle; presynaptic; fluorescence SYNAPTIC VESICLES are recycled in nerve terminals via a variety of different endocytic pathways, each potentially using a different subset of the total endocytic machinery (Voglmaier and Edwards

(2014) RGK protein-mediated impairment of slow depolarization-dependent Ca 2+ entry into developing myotubes, Channels, 8:3,[243][244][245][246][247][248]