The Study On Phosphorylation And Structure-Function Relationship Of Voltage-gated Sodium Channels

Voltage-gated sodium channels (VGSCs) are key molecules contributing to theinitiation of actional potentials, playing an indispensible role in electric excitationand firing of neurons. As transmembrane proteins with conserved structure, VGSCsare subjected to dynamic modulations arising from both extra-and intracellularfactors. Meanwhile, VGSCs are targetted by numerous natural neurotoxns orsynthesized compands through blocking or modulating the ion permeable selectivityand shifting the structure-function balance of channel gating,which result in clinicalpathology or relief from severe syndromes.By means of electrophysiological andmolecular biological methods, this study investigated the regulation of VGSCs byendogenous phosphorylation and functional modulation by site-4VGSC modulatorfrom Buthus martensii Karsch (BmK).The main focus of this researchinclude:1.Structural and functional regulation of VGSCs by PKA phosphorylation;2.The subtype selectivity,pharmacological efficacy of site-4modulators on VGSCsand underlying molecular mechanisms.Part1: PKA phosphorylation have been considered to involved in overallregulation circuit of VGSCs whose activities are varied with tissue-specific subtypes,membrane potential and divergent intra-/extracellular environment.In context withphosphorylation or other regulatory factors,VGSCs are deemed to participatedirectly or indirectly in many physiological/pathological events,such as convergentneuronal transmitting, synaptic plasticity and cell signalling.This research found thatPKA activation could suppress peak currents of VGSCs without any impact ongating properties.The modulation of10nM BmK AS on VGSC inactivation couldbe further enhanced by PKA phosphorylation.Comparatively, the modulation of1nM or100nM BmK AS on VGSCs were alieviated after PKAactivation.Pre-treatment with Isoproterenol, a β2-adrenergic receptor agonist which could elevated intracellular cAMP level, disrupted the modulation of BmK AS onvoltage-dependent inactivation to a reverse direction. Thus, PKA activation wasthought to remove the ‘U shape’ dose-dependent effects of BmK AS. By localizationof the interaction sites of BmK AS on VGSC, it was predicted that the receptor siteof BmK AS and phosphorylation sites of PKA on VGSC might be conformationallyapproximate, which indicate a potential interaction between these twomolecules.This study may provide clues for understanding the role of PKAphosphorylation in VGSC-mediated pharmacology.Part2：BmK IT2is regarded as the receptor site4-specific modulator ofsodium channels with the depressant anti-insect toxicity and antinociceptive oranticonvulsant activity, but the underlying molecular mechanism remains to beclarified. In this study, the selectivity of BmK IT2among four sodium channelsubtypes expressed in Xenopus oocytes was assessed, and the binding localization ofBmK IT2on receptor site4of voltage-gated sodium channels was determined usingtwo-electrode voltage clamp recording. Results showed BmK IT2could stronglyhyperpolarize the activation of DmNav1, an insect VGSC from Drosophila Para, buthardly affect rNav1.2, rNav1.3and mNav1.6, three mammalian central neuronalsodium channel subtypes. By replacing each domain (DI-DIV) of rNav1.2with thatof DmNav1, it was found that BmK IT2-specificity for DmNav1could be conferredby DIII while the interaction with channel voltage-sensor was mediated throughbinding to DII. Analysis of subsequent DmNav1mutants further highlighted that, inaddition to the contribution of Glu896, Leu899, Gly904in DII S4for functional actionof BmK IT2, residues in DIII pore loop, esp. Ile1529and Arg1530were critical forrecognition and binding of BmK IT2. The data found in the study revealed twonon-interacting regions, comprising DII and DIII on DmNav1, played separated butindispensable roles in the interaction with BmK IT2. The insensitivity ofmammalian central neuronal sodium channel isoforms to BmK IT2might suggestthe other mechanism or targeted subtypes involved in the suppressive activity of BmK IT2in pathological models.