Biophysical characterization of neuronal and skeletal muscle sodium channels, and their regulation by auxiliary beta subunits

Authors: Zhao, Juan
Advisor: Chahine, Mohamed
Abstract: Voltage-gated Na channels are responsible for the rising phase of action potentials, and consist of a pore-forming α subunit and one or more auxiliary β subunits. The α subunit alone is sufficient for the functional expression of Na channels, however, β subunits modulate the location, expression and functional properties of α subunits. My thesis will focus on three neuronal Na channels (Nav1.6, Nav1.7 and Nav1.8) and one skeletal muscle Na channel (Nav1.4). Neuronal Na channel are key players in the impulse propagation along axon. Nav1.7 and Nav1.8 are the main Na channels expressed in DRG neurons, and their altered expression and modulation following injury and inflammation play a major role in nociception and chronic pain. Nav1.6 is highly concentrated at nodes of Ranvier, and has a critical role not only in saltatory conduction but also in high-frequency repetitive firing. Skeletal muscle Na channel Nav1.4 is the initiator of muscle contraction. Mutations in Nav1.4 cause skeletal muscle channelopathies. Guiding questions for our investigations were: 1) How do auxiliary β subunits regulate peripheral nerve Na channel Nav1.6 and Nav1.8? 2) What is the underlying biophysical defect of M1476I, a novel founder SCN4A mutation associated with painful cold-induced myotonia in French Canadians? 3) What is the biophysical characterization of the Nav1.6 persistent current? 4) What is the expression pattern of auxiliary  subunits, and how do β subunits regulate Nav1.7 in DRG neurons? We addressed these questions by multiple approaches including patch clamp techniques for whole-cell and single-channel recordings in heterologous expression systems; immunohistochemistry, single-cell RT-PCR and immunoprecipitation in DRG neurons. Firstly, we employed single-cell RT-PCR of acutely dissociated DRG neurons to identify the expression of β1-4 subunits in small-diameter sensory neurons. Our results indicated that small-diameter DRG neurons widely expressed Nav1.6 and Nav1.8 channels and β1-β3 subunits. Co-expression studies were used to assess the regulation of Nav1.6 and Nav1.8 by β subunits. The β1 subunit induced a significant increase in the current density of Nav1.8 when co-expressed in HEK293 cells, but had no effect on that of Nav1.6. In addition, the C-terminal domain of β1 was involved in the modulation of Nav1.8 channel based on the results of experiments with β1/β2 chimeras harboring various regions of the strongly regulating β1 together with the weakly regulating β2 subunit. Secondly, we investigated the biophysical defects of M1476I mutation in Nav1.4 channels using whole-cell patch-clamp technique in tsA201 cells. M1476I mutant channel exhibited similar biophysical defects compared with other PAM-causing mutations, including an increased persistent current of Nav1.4, a slower current decay, a positive shift of fast inactivation, and an accelerated recovery from fast inactivation. Lowering the temperature slowed the kinetics for both wide-type and mutant channels, and worsened the defective fast inactivation of M1476I channels by further increasing the amplitude of the persistent current. Mexiletine helps relieve myotonia in M1476I carriers by effectively suppressing the increased persistent current, except for the use-dependent block. However, mexiletine had a reduced effectiveness on the use-dependent block of M1476I channels, and that was associated with a faster recovery from mexiletine block of mutant channels. Thirdly, we characterized the whole-cell and single-channel properties of Nav1.6 persistent currents expressed in HEK293 cells. We noted that Nav1.6 persistent current was highly sensitive to the composition of the internal solution, and persistent current was rarely detectable when CsF instead of CsCl was used. By substituting CsF for CsCl in the intracellular solution, we showed that Nav1.6 persistent current in the whole-cell configuration was 3–5% of the peak transient current. This amplitude of persistent current was similar to the ratio between peak and persistent open probability observed in the single-channel recording, indicating that the occurrence of late channel reopenings accounts for the persistent macroscopic Na current typical of Nav1.6. Finally, we employed a combination of single-cell RT-PCR, immunocytochemistry and immunoprecipitation to investigate  subunit expression in subpopulations of sensory neurons.  subunits were differentially expressed in small (2, 3) and large (1, 2) DRG neurons. Nav1.7 mRNA was significantly co-expressed with the 2 and 3 subunits in the same population of small-diameter DRG neurons. They formed stable protein-protein interactions and co-localized within the plasma membranes of neurons.When co-expressed in HEK293 cells, 3 and 1 subunits shifted activation and inactivation curves respectively and induced a marked increase in Nav1.7 window current. Our data indicated a preferential expression of  subunits in small and large DRG neurons and a subunit-specific Nav1.7 regulation in these subpopulations of sensory neurons.
Document Type: Thèse de doctorat
Issue Date: 2012
Open Access Date: 18 April 2018
Grantor: Université Laval
Collection:Thèses et mémoires

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