The Molecular Interaction Basis Of Voltage-gated Sodium Channel With Peptide Toxins From The Spider Chilobrachys Jingzhao

The bioactive peptide toxins isolated from the spiders can selectively affects different kinds of molecular targets, such as voltage-gated sodiumchannel expressed on the cell membrane.On the base of its high selectivity and efficiency, the bioactive peptide toxins isolated from the spiders can be used as molecular probe to do research on its molecular targets,such as voltage gated sodium channel, or be developed into useful drugs with prosperous market,such as Zinotide. Chilobrachys jingzhao is a kind of brand new spider discovered in the province of Guangxi,which belongs to Selenocosmiinae. Jingzhaotoxin-V(JZTX-V) is a29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao, which has an inhibitory effect on TTX-S and TTX-R sodiumcurrents on rat DRG cells with IC50values of27.6and30.2nM, respectively, and is able to shift the activation and inactivation curves tothe depolarizing and the hyperpolarizing direction, respectively.First, in this thesis,we shed great light on the structure and function of JZTX-V, proving that JZTX-V has a much stronger inhibitory effect on Nav1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC50value of5.12nM, stronger than its inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG In our experiment, we chose to investigate in more detail the ability of JZTX-V to block currents generated by four human (h) or rat (r) VGSC subtypes (hNaV1.3, rNaV1.4, hNaV1.5, and hNaV1.7) expressed in HEK human embryonic kidney (HEK)293cells.The result shows that Nav1.4, a skeletal muscle subtype preferentially expressed in skeletal muscle cells, is the most sensitive to JZTX-V among the four tested VGSC isoforms,while JZTX-V has the weakest inhibitory effect on Nav1.5. Fitting the data with the Hill equation yielded the following IC50values:hNav1.3,292±61nM; rNav1.4,5.12±0.87nM, hNav1.5,2.7±0.779μM and hNav1.7,61.7±1.2nM. The time constants of inhibition were34.01±6.81s(rNav1.4),133.33±27.96s (hNavl.7),48.54±11.35s (hNav1.3) and34.24±8.13s(hNavl.5), respectively.JZTX-V have strong inhibitory effect on the peak current of Navl.4, while JZTX-V has not obvious effect on steady-state activation and inactivation of the residual currents conducted by skeletal muscle VGSCs, but it can change the slope factor of steady-state activation and inactivation curves. We tried to investigate and disclose the amino acid residues of JZTX-V that play a crucial function in binding to rNaV1.4by introducing site mutations into every amino acid position except for cysteine, but we were able to mutate only19residues successfully. On the one hand, compared to recombinant JZTX-V, alanine mutations of3hydrophobic residues (W5A, M6A, and W7A) were able to alter toxin affinity toward Nav1.4significantly. Fitting a Hill equation to the data yielded IC50values of0.313±0.26(W5A),0.252±0.45(M6A), and1.92±0.31μM (W7A), suggesting that the three mutants reduce JZTX-V binding affinity by61-,49-and375-fold, respectively. In particular, the W7A mutation nearly abolished the binding affinity of JZTX-V on rNav1.4. On the other hand, compared to wild type JZTX-V, alanine mutations of two positively charged residues (R20A and K22A) were able to alter toxin affinity toward NaV1.4significantly and yielded IC50values of2.1±0.28(R20A) and0.659±0.011(K22A) μM, respectively. These results show that the mutations of these two positively charged residues were able to reduce JZTX-V binding affinity by410-and128-fold, respectively. JZTX-V adopts a Janus-faced surface profile with two functional bioactive faces, in which a large hydrophobic patch consisting of three hydrophobic residues (W5, M6and W7) is formed on the surface, and two positively charged residues (R20and K22,) surround the hydrophobic patch. Previous phospholipid membrane-binding experiments indicated that the basic residues on the surface of JZTX-V play a critical role in the binding to VGSC,which has been verified in this thesis. Next, we mutated the61amino acid residues located in extracellular linkers of domain I-IV in order to identify the interaction site on VGSC subtype Nav1.4, but we found these amino acid residues has no obvious effect on the interaction between Nav1.4and JZTX-V, which means the interaction sites are not located in this area and rules out the possibility that JZTX-V binds to the extracellular linker of voltage gated sodium channel.Second, this thesis also designs experiments to investigate whether β1subunit has effect on the interaction between JZTX-V and VGSC subtype Nav1.4. First, the results show that β1subunit is able to modify the dynamic features of Nav1.4. Second, the results reveal that β1subunit has the capacity to block the inhibitory effect of JZTX-V on Navl.4.Third, in this thesis, electrophysiological studies on mutant rNav1.4R222W, which leads to hypokalaemic periodic paralysis (HypoPP) in a16years old boy, was conveyed in great detail. Hypokalaemic periodic paralysis is a dominantly inherited muscle disorder characterized by episodes of flaccid weakness. The results show that, compared to the wild type rNav1.4, rNav1.4R222W is able to shift the steady-state activation and inactivation curves to depolarizing (-6mV) and hyperpolarizing (-5mV), respectively. Patch-clamp recordings show an enhanced fast inactivation for this mutant, which may reduce the number of functional sodium channels at resting membrane potential and contribute to the long-lasting periods of paralysis experienced by hypokalaemic periodic paralysis patients. The gating of this mutant, rNav1.4R222W, cannot be modulated by changes of extracellular pH, which may explain why there is no therapeutic effect of drug acetazolamide on this kind hypokalaemic periodic paralysis patients. In conclusion, this thesis is able to reveal the gating mechanism of the hypokalaemic periodic paralysis mutant Nav1.4R222W, as well as why there is no therapeutic effect of drug acetazolamide on this kind hypokalaemic periodic paralysis patients. Meanwhile, in the thesis, we are able to prove that JZTX-V is an antagonist of voltage gated sodium channel subtype Nav1.4, which means it can be used as a molecular probe for investigation or be used as a potential drug molecule for muscle disease.