Differential Recording of Voltage Changes at the Surface and Transverse Tubular System Membranes of Mammalian Skeletal Muscle Fibers using Di-8-Anepps and Global and TIRFM

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Aiming to investigate the distribution of ClC-1 and KIR channels at the sarcolemma and transverse tubular system (TTS) membranes of mammalian skeletal muscle fibers, we used global and total internal reflection fluorescence microscopy (TIRFM) to monitor voltage changes in these compartments, respectively. Enzymatically-dissociated fibers from murine FDB and interosseus muscles were stained with the potentiometric dye di-8- ANEPPS, and voltage-clamped with a two-microelectrode system. Ion substitutions were used to isolate and characterize the specific ClC-1 (ICl) and KIR (IKIR) currents: 70 mM internal [Cl-] and 120 mM external [K+], respectively. Also, 9-ACA and TEA were used to, respectively, block these currents. Global di-8-ANEPPS signals report, early after the onset of large hyperpolarizing pulses, ICl-dependent attenuations with respect to those recorded in the presence of 9-ACA. Peak attenuation levels of ∼35% were observed for ICl of ∼900 µA/cm2. Large attenuations were similarly observed in global signals recorded the presence of large IKIR's with respect to those in TEA. In contrast, TIRFM di-8-ANEPPS signals demonstrate only minor current-dependent attenuations (<10%) under conditions in which global signals evidenced much larger attenuations. Overall, our results demonstrate that voltage changes at the TTS membranes display prominent current-dependent attenuations while the sarcolemma is largely under voltage-clamp control. A radial cable model of the TTS, including equations for each current pathway and luminal accumulation/depletion of ions, was used to quantitatively predict the ionic currents and to assess their effects on average TTS voltage changes. Comparative analysis of global optical data with model predictions of voltage changes in the TTS suggests that both ClC-1 and KIR channels are equally distributed in both membrane compartments. Supported by NIH grants AR047664, AR041802, and AR054816.