Dataset Information

Rat Brain LC-MS/MS - Quantitative Proteomics Reveals Protein-Protein Interactions with Fibroblast Growth Factor12 as a Component of the Nav1

ABSTRACT: Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials in excitable cells. While the channel is functional on its own, it is the transient and stable protein-protein interactions that modulate functional outcomes. AP-MS has been successfully applied to a number of ion channels. However to the best of our knowledge, no AP-MS study has been carried out on any member of the voltage-gated sodium channel family.

INSTRUMENT(S): LTQ Orbitrap Elite

TISSUE(S): Brain, Electrically Active Cell

SUBMITTER: Norelle Wildburger

LAB HEAD: Carol Lynn Nilsson

PROVIDER: PXD000719 | Pride | 2015-03-03

REPOSITORIES: Pride

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Quantitative proteomics reveals protein-protein interactions with fibroblast growth factor 12 as a component of the voltage-gated sodium channel 1.2 (nav1.2) macromolecular complex in Mammalian brain.

Wildburger Norelle C NC Ali Syed R SR Hsu Wei-Chun J WC Shavkunov Alexander S AS Nenov Miroslav N MN Lichti Cheryl F CF LeDuc Richard D RD Mostovenko Ekaterina E Panova-Elektronova Neli I NI Emmett Mark R MR Nilsson Carol L CL Laezza Fernanda F

Molecular & cellular proteomics : MCP 20150227 5

Voltage-gated sodium channels (Nav1.1-Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein-protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here w ...[more]

PMID: 25724910

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Project description:Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1-R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.

Project description:Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Nav?4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Nav?4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Nav?4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Nav?4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Nav?4 peptide. Although the Nav?4 peptide substantially increased the rate of recovery from apparent inactivation, Nav?4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.

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	Norelle-NAV-20Aug2013.mgf	Mgf
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