The New Mechanism Of The Interactions Between Peptides And Potassium Channels

Potassium channels are widely distributed membrane proteins which are closely related to various physiology and pathophysiology functions, such as membrane potential, muscle contraction, secretion, volume regulation and cell proliferation. A lot of peptides which are from isolation and purification, genetic engineering and design serve as indispensably tools and play important roles in the structure-functional research of potassium channels. Due to the challenge of resolution of peptide-potassium channel complex structure, the mechanism of peptide-channel interactions progress slowly. In combination with the experimental techniques of molecular biology, cell biology, electrophysiology and bioinformatics, we focused on the novel mechanisms of peptide-channel interactions, which will further accelerate research and application of peptide-potassium channel interactions.Among a lot of scorpion toxins acting on potassium channels, only a limited subset was capable to selectively block small-conductance calcium-activated potassium (SK) channels while most classical scorpion toxins (such as Charybdotoxin (ChTX)) could not bind to SK channels. The structural bases of this selective SK channel recognition remain unclear. In this work, scorpion toxin BmP05was used as a molecular tool. We found SK-selective toxins exhibited no more than three basic amino acid residues in their channel-binding interfaces. If one or two basic residues were added to the binding interface of BmP05,100nM mutants could only inhibited8.7-30.5%of the SK3channel currents. When came to the SK3channels, we demonstrated the key role of the electric charges of two conserved arginine residues (R485and R489) from the SK3channel outer vestibule in the selective recognition. Indeed, individually substituting these residues by histidyl or lysyl (maintaining partially or fully the positive electric charge), while decreasing the affinity of BmP05, still preserved its toxin-sensitivity profile (as evidenced by the lack of recognition by many other potassium channel-sensitive ChTX). In contrast, when R485or R489residue of SK3channel was mutated towards an acidic (E) or alcoholic (S) amino acid residue, the channel lost its sensitivity to BmP05and became susceptible to a'new' blocking activity by insensitive scorpion toxin ChTX. Besides these basic SK3channel residues important for sensitivity, two acidic D492and D518residues, also located in the SK3channel outer vestibule, were identified as being critical for toxin affinity. Further, molecular modeling data indicated the existence of two basic rings (called "peptide screener"), which was formed by R485and R489residues and stabilized by strong ionic interactions with the acidic D492and D518residues, respectively. In conclusion, the unique "peptide screener" restricted the type of SK-selective toxins by spatial orientations and electrostatic repulsion forces, which illuminated the structural basis of selective SK3channel recognition by scorpion toxins.At present, the SK-selective scorpion toxins (scyllatoxin, BmP05and so forth) mainly use a helix as their active epitope when binding to SK channels. A designed potassium channel inhibitor, BmP05-T, used the (3-sheet domain as the binding interface, with the IC50value of22.01nM for Kv1.3channels. The pharmacological experiments further revealed that BmP05-T still preserved the binding affinity for SK channels with the IC50value of130.6nM, which implied a novel mechanism of BmP05-T-SK3channels interactions. In order to investigate their interaction mechanism, we respectively replaced the basic residues in the a helix domain and ? sheet domains of the scorpion toxin BmP05-T by alanine. The pharmacological data showed that the affinity of BmP05-T-K6A, BmP05-T-R7A and BmPO5-T-R13A decreased about6fold while the affinity of BmP05-T-K20A, BmP05-T-K25A, BmP05-T-K27A decreased more than11fold. It was also found that1?M BmP05-T-K27A mutant only blocked about8.40%SK3channel currents. All these results suggested the scorpion toxin BmP05-T used ? sheet as the binding interface for interaction with the SK3channels. Combined with the3D structure resolution of BmP05-T and molecular modeling of BmP05-T-SK3channel interaction, we found that the side chain of K27in the ? sheet domain of the scorpion toxin BmP05-T was used to plug into the selective filter while K6, R7and R13in the a helix domain kept away from the pore region of SK3channels. In summary, the structural characters of the peptide-channel interaction illuminated the novel binding interface of ? sheet domain was used when the scorpion toxin BmP05-T recognized the SK3channels. These results not only enriched the knowledge of new binding interface of SK channel selective peptides, but also provided the possibility of the design of new blockers that display selectivity between SK channel subtypes.The human potassium channels are widely inhibited by exogenous peptide toxins (scorpion toxins, snake toxins, sea anemone toxins and so forth). However, it remains a mystery whether there is any human endogenous potassium channel inhibitor. Based on the structural commonness between Kvl.3channel selective toxins and human ?-defensin2(hBD2), we demonstrated that1?M hBD2could respectively inhibit about16.50%,95.71%and12.53%of Kv1.2, Kv1.3and KCNQ1channel currents while it showed no effect on IKCa, SKCa3and hERG channels currents. On the basis of the concentration-response curve for Kvl.3channels, the IC5o value of hBD2binding was22.07pM. By constructing the chimeras between Kv1.2and Kv1.3channels, we found that hBD2bound not only S5-S6linker region but also S1-S2linker of Kvl.3channel. Similar to the classical toxin blockers, the outer pore domain of Kvl.3channel was found responsible for hBD2recognition through the mutagenesis experiments. Furthermore, hBD2could also bind to functional sites on the S1-S2linker of Kvl.3channel and counteract the movement of the S4helix by electrostatic repulsion forces, thereby changing the gating dynamics of Kv1.3channel. In summary, we firstly reported the human endogenous potassium channel inhibitor which could modulate the Kvl.3channel gating by binding to S1-S2linker. These results hinted more endogenous peptide inhibitors of potassium channels and pave the promising avenue to investigate novel functions of these peptides.In conclusion, we paid our attention to the novel molecular mechanisms of the interaction between peptides and potassium channels. Firstly, we found a special "peptide screener" in SK3channel, which controls the selective recognition by peptides; Secondly, we showed the binding properties of a designed peptide, BmP05-T, which was firstly reported to use the ?-sheet as the binding interface when interacted with SK channels; Finally, we found the novel functions and mechanism when endogenous hBD2interacted with Kvl.3channel. Our results enriched the diversity of the mechanisms of peptide-potassium channel interaction and will lead to the development of specific peptide modulators and therapeutic drugs which selectively target potassium channels.