Browsing School, Graduate by Title "Optical Recordings of Action Potentials and Voltage Sensing Domains"
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Optical Recordings of Action Potentials and Voltage Sensing DomainsExcitation-contraction coupling (ECC) allows muscle to translate an action potential (AP) into muscle contraction. Optical methods for measuring membrane potential changes can expand our understanding of excitable cell function. Applying the potentiometric dye Di-8-ANEPPS and high-speed confocal microscopy to flexor digitorum brevis (FDB) muscle fibers from adult mice we non-invasively measure the electrical properties of these fibers. We determined action potential conduction velocity by comparing the time course of action potentials initiated at either end of muscle fibers by using alternate polarity electric field stimulation. Action potentials propagated longitudinally at a velocity of 0.39 ± 0.02 m/s. Conduction velocity of calcium transients, using mag-fluo-4, a low-affinity calcium indicator, was 0.37 ± 0.03 m/s, similar to Di-8-ANEPPS. We used mag-fluo-4 to examine whether our approach could capture conduction changes due to ionic concentrations, fiber length, and a lack of dystrophin, and found that we could. A lumped component equivalent electrical circuit model of the muscle fiber’s passive properties reproduced the observed passive responses of muscle fibers. This method using dyes allows the study the action potential propagation in a non-invasive manner in FDB fibers under differing physiological conditions and in various disease states. In skeletal muscle, AP sensing is governed by Cav1.1. Cav1.1 has 4 voltage sensor domains (VSDs) located in the membrane that that move in response to changes in membrane potential. During an AP these VSDs shift, initiating calcium release. We have not yet elucidated VSD movement in Cav1.1. Here we transfect mouse FDBs with a version of Cav1.1 that has a cysteine near the VSD. This allows us to attach a thiol-reactive fluorescent probe near the VSD and track its movement. This did not noticeably affect trafficking or function of Cav1.1. In resting conditions, our cysteine of interest should be embedded in the membrane. Upon depolarization, this cysteine shifts out of the membrane, allowing us to detect its movement as a change in fluorescence after changes in fluorophore environment. Using field stimulation fluorometry, we observed the shifts of Cav1.1 VSDs in response to field stimulation and analyzed how they correspond to Ca2+ release in native tissue.