The Pro-apoptotic Protein Bax Derived Peptide Potently Inhibits The Voltage-gated Potassium Channels

The Pro-apoptotic protein Bax(BCL-2 associated X protein)acts on the mitochondrial voltage-gated potassium channel subtype Kv1.3(mito Kv1.3)to induce cell apoptosis by affecting mitochondrial membrane potential and inducing ROS(reactive oxygen species)and cytochrome C release.The designation of the present study was as follows:i)design a Baxderived peptide(BaxP1)which contains the sequence from the begining of H?5 to the end of H?6 helixes in Bax,and two cysteines at the end of these two helixes which make it a cyclopeptide,(sequence:CGNWGRVVALFYFASKLVLKALCTKVPELI RTIMGWTLDFLRERLGC;the underlined part is the Baxpeptide sequence).ii)produce the BaxP1 peptide as fusion protein with the glutathione S-transferase(GST-BaxP1)and test its activity on Kv1.3channel;iii)shorten the BaxP1 peptide as much as possible to make a voltage-gated potassium channels(Kvs)inhibitory module with the minimal size,this module might be useful in designing Kv1.3 specific antagonist.To achieve this goal,we first optimized and synthesized the BaxP1 coding sequence according to the codon usage preference of E.coli,and constructed the prokaryotic expression vector pEGX-4T-2-BaxP1.Furthermore,four BaxP1 mutants expression vectors(pEGX-4T-2-BaxP1/K30E,pEGX-4T-2-BaxP1/C6A,pEGX-4T-2-BaxP1/C52A,pEGX-4T-2-BaxP1/C6A-C52)were constructed by site-directed mutagenesis.These five recombinant plasmids were transformed into E.coli BL21(DE3)strain and protein expression was induced by IPTG.SDS-PAGE analysis showed that all of the five recombinant proteins were abundantly expressed in soluble form,and they were purified to homogeneity by using Glutathione sepharose4B affinity medium and RP-HPLC purification(GST-BaxP1,GST-BaxP1/K30E,GST-BaxP1/C6A,GST-BaxP1/C52A,GST-BaxP1/C6A-C52A).We test the activity of these five recombinant proteins on voltage-gated potassium and sodium channels(Kvs and Navs)by using whole-cell patch clamp recordings.The result showed that GST-BaxP1strongly inhibits Kv1.3 current but not Navs currents heterologously expressed in HEK293T cells with a IC₅₀of approximately 230 n M,and the GST tag protein did not affect Kv1.3 currents even at high dose.Testing the activity of GST-BaxP1 on various Kvs(including Kv1.1,Kv1.4,Kv1.5,Kv2.1,Kv3.1 and Kv4.1)showed that it was a non-selective kvs inhibitor,and the IC₅₀values ranged from 300 n M to700 n M.However,the GST-BaxP1/K30E,GST-BaxP1/C6A,GST-BaxP1/C52A and GST-BaxP1/C6A-C52A mutant recombinant proteins did not affect Kv1.3 currents.It has been reported that mutating the 128th lysine to glutamine in Bax(K128E)make it function-loss in inhibiting Kv1.3.The K30 in GST-BaxP1 is analogous to K128 in Bax,thus the result highlighted the role of this positive charged lysine residue in inhibiting Kv1.3 not only in Baxprotein but also in Baxderived peptide.More importantly,the disulfide bond in BaxP1 should play a crucial role in maintaining the conformation and activity of the peptide as destroying it by mutation(GST-BaxP1/C6A?GST-BaxP1/C52A and GST-BaxP1/C6A-C52A)make the peptide inactive.The follow-up study will work on shortening the BaxP1 peptide as much as possible to make a Kvs inhibitory module for Kv1.3 specific antagonist designation.