functional-site-directed-fluorometry-native-cells-to-study-skeletal

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, ...

University of Maryland School of Medicine

The skeletal muscle L-type Ca channel (Ca1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca release. Ca1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca release remains unknown. To investigate the role of VSDs in excitation-contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of Ca1.1, expressed each construct via in vivo gene transfer electroporation, and used  AP fluorometry to track the movement of each Ca1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of Ca1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels' VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca, and the calculated rate of Ca release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca release, and thus could be involved in voltage sensor rearrangements or Ca release activation.

Voltage sensor movements of Ca1.1 during an action potential in skeletal muscle fibers.

Ca influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca influx, to mediate disparate functions is diversity of HVACC pore-forming α and auxiliary Caβ-Caβ subunits. Selective Caα blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on Caβ isoform is non-existent. Conventional gene knockout/shRNA approaches do not adequately address this deficit owing to subunit reshuffling and partially overlapping functions of Caβ isoforms. Here, we identify a nanobody (nb.E8) that selectively binds Caβ SH3 domain and inhibits Caβ-associated HVACCs by reducing channel surface density, decreasing open probability, and speeding inactivation. Functionalizing nb.E8 with Nedd4L HECT domain yielded Chisel-1 which eliminated current through Caβ-reconstituted Ca1/Ca2 and native Ca1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca influx and excitation-transcription coupling in hippocampal neurons, but was inert against Caβ-associated Ca1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of Caβ signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties.

Selective posttranslational inhibition of Caβ-associated voltage-dependent calcium channels with a functionalized nanobody.

The Ca1.1 voltage-gated Ca channel carries L-type Ca current and is the voltage-sensor for excitation-contraction (EC) coupling in skeletal muscle. Significant breakthroughs in the EC coupling field have often been close on the heels of technological advancement. In particular, Ca1.1 was the first voltage-gated Ca channel to be cloned, the first ion channel to have its gating current measured and the first ion channel to have an effectively null animal model. Though these innovations have provided invaluable information regarding how Ca1.1 detects changes in membrane potential and transmits intra- and inter-molecular signals which cause opening of the channel pore and support Ca release from the sarcoplasmic reticulum remain elusive. Here, we review current perspectives on this topic including the recent application of functional site-directed fluorometry.

Advances in Ca1.1 gating: New insights into permeation and voltage-sensing mechanisms.

In skeletal muscle, Ca 1.1 serves as the voltage sensor for both excitation-contraction coupling (ECC) and L-type Ca channel activation. We have recently adapted the technique of action potential (AP) voltage clamp (APVC) to monitor the current generated by the movement of intramembrane voltage sensors (I ) during single imposed transverse tubular AP-like depolarization waveforms (I ). We now extend this procedure to monitoring I , and Ca currents during trains of tubular AP-like waveforms in adult murine skeletal muscle fibers, and compare them with the trajectories of APs and AP-induced Ca release measured in other fibers using field stimulation and optical probes. The AP waveform remains relatively constant during brief trains (<1 sec) for propagating APs in non-V clamped fibers. Trains of 10 AP-like depolarizations at 10 Hz (900 ms), 50 Hz (180 ms), or 100 Hz (90 ms) did not alter I amplitude or kinetics, consistent with previous findings in isolated muscle fibers where negligible charge immobilization occurred during 100 ms step depolarizations. Using field stimulation, Ca release did exhibit a considerable decline from pulse to pulse during the train, also consistent with previous findings, indicating that the decline of Ca release during a short train of APs is not correlated to modification of charge movement. Ca currents during single or 10 Hz trains of AP-like depolarizations were hardly detectable, were minimal during 50 Hz trains, and became more evident during 100 Hz trains in some fibers. Our results verify predictions on the behavior of the ECC machinery in response to AP-like depolarizations and provide a direct demonstration that Ca currents elicited by single AP-like waveforms are negligible, but can become more prominent in some fibers during short high-frequency train stimulation that elicits maximal isometric force.

Article	Year
Voltage sensor movements of Ca1.1 during an action potential in skeletal muscle fibers. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 34583989	2021
Selective posttranslational inhibition of Caβ-associated voltage-dependent calcium channels with a functionalized nanobody. Nature communications\| PubMed ID: 36494348	2022
Advances in Ca1.1 gating: New insights into permeation and voltage-sensing mechanisms. Channels (Austin, Tex.)\| PubMed ID: 36642864	2023
Voltage sensor current, SR Ca release, and Ca channel current during trains of action potential-like depolarizations of skeletal muscle fibers. Physiological reports\| PubMed ID: 37147904	2023

Hugo Bibollet

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