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.

P/Q-type Ca currents mediated by Ca2.1 channels are essential for active neurotransmitter release at neuromuscular junctions and many central synapses. Mutations in CACNA1A, the gene encoding the principal Ca2.1 α subunit, cause a broad spectrum of neurological disorders. Typically, gain-of-function (GOF) mutations are associated with migraine and epilepsy while loss-of-function (LOF) mutations are causative for episodic and congenital ataxias. However, a cluster of severe Ca2.1 channelopathies have overlapping presentations which suggests that channel dysfunction in these disorders cannot always be defined bimodally as GOF or LOF. In particular, the R1667P mutation causes focal seizures, generalized hypotonia, dysarthria, congenital ataxia and, in one case, cerebral edema leading ultimately to death. Here, we demonstrate that the R1667P mutation causes both channel GOF (hyperpolarizing voltage-dependence of activation, slowed deactivation) and LOF (slowed activation kinetics) when expressed heterologously in tsA-201 cells. We also observed a substantial reduction in Ca current density in this heterologous system. These changes in channel gating and availability/expression manifested in diminished Ca flux during action potential-like stimuli. However, the integrated Ca fluxes were no different when normalized to tail current amplitude measured upon repolarization from the reversal potential. In summary, our findings indicate a complex functional effect of R1667P and support the idea that pathological missense mutations in Ca2.1 may not represent exclusively GOF or LOF.

Complex effects on Ca2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder.

P/Q-type Ca flux into nerve terminals  Ca2.1 channels is essential for neurotransmitter release at neuromuscular junctions and nearly all central synapses. Mutations in , the gene encoding Ca2.1, cause a spectrum of pediatric neurological disorders. We have identified a patient harboring an autosomal-dominant  frameshift-causing nucleotide duplication in  (c.5018dupG). The duplicated guanine precipitated 43 residues of altered amino acid sequence beginning with a glutamine to serine substitution in Ca2.1 at position 1674 ending with a premature stop codon (Ca2.1 p.Gln1674Serfs*43). The patient presented with episodic downbeat vertical nystagmus, hypotonia, ataxia, developmental delay and febrile seizures. In patch-clamp experiments, no Ba current was observed in tsA-201 cells expressing Ca2.1 p.Gln1674Serfs*43 with β and αδ-1 auxiliary subunits. The ablation of divalent flux in response to depolarization was likely attributable to the inability of Ca2.1 p.Gln1674Serfs*43 to form a complete channel pore. Our results suggest that the pathology resulting from this frameshift-inducing nucleotide duplication is a consequence of an effective haploinsufficiency.

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
Complex effects on Ca2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder. Scientific reports\| PubMed ID: 35655070	2022
A neurodevelopmental disorder caused by a dysfunctional allele. eNeurologicalSci\| PubMed ID: 36938367	2023

Daniel F. Bennett

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