Interaction Sites Between The MSlo1 Pore And The NH2 Terminus Of The Beta2 Subunit, Probed With A Three-residue Sensor

Calcium- and voltage- activated large conduction potassium channel (Maxik channel, also termed BK channel) extensively exists in excitable cells, especially in neural system. BK channel plays a significant physiological role in mediating the concentration of intracellular calcium ions and the membrane potential. At present, the voltage dependent potassium channel (Kv channel) has been deeply studied. BK channel shares structural and functional similarities with Kv channel and also has its own characteristics. For example, they all can combine auxiliaryβsubunits, and all have N-type inactivation induced by the hydrophobic amino acids of the N terminals. However, unlike Kv channels, the intracellular blockers (such as TEA and QX-314) of BK channels can not slow the process of inactivation. Therefore, how does theαsubunit of BK channel interact with theβsubunit during inactivation, and what are they interaction sites, these questions are still unsolved since being presented.We mutated the first three amino acids of hβ2 N terminal FIW into FWI, found that FWI mutant had no impact on the inactivation ofαsubunit, but its time curve of recovery was changed into bi-exponential characteristic from mon-exponent. Utilizing this feature of FWI, we mutated the hydrophobic amino acids in the pore formed S6 segment ofαsubunit into less hydrophobic alanine. The interaction sites betweenαsubunit andβ2 subunit in the process of inactivation were detected by the coexpression of S6 mutants and FWI.The main research achievements in this study are as follows:(1) Among the S6 mutants, mSlo1-I323A coexpressed with hβ2-FWI, their slow component of the bi-exponent was maximally reduced. That is, its recovery curve can be almost fitted into mon-exponent. It demonstrated that I323 is the main interaction site withβ2 subunit during the inactivation. In additional, the fast components of M314A and V319A were extrodinarily faster, sugesting that the two points have certain effect in the process of inactivation. Based on the linear structure relationship of FWI, we inferred the following interaction relationship: I323-I, V319-W, M314-F; I323 played the leading role.(2) Although the sensitivity of V319A to QX-314 is higher than other sites, there is no specific binding site for QX-314 in the pore. We presented one step non-competition model. For Ile-323 is the last residue in the pore, the interaction site ofβ2 inactivation domain is on the entrance of the pore, the interaction sites of QX-314 are in the pore, therefore, QX-314 would not impact the process of inactivation. This model explained that why intracellular blockers do not slow the inactivation process of BK channel. We provided important experimental and theoretical support for understanding the structure and the gating mechanism of BK channel.(3) I323A had outward rectification phenomena in both single channel level and microcurrent level, but wide type mSlo1 did not; the single channel current of I323A is flickery, like the single channel current of dSlo1A. These demonstrated that Ile-323 can also mediate the gating of BK channel. The corresponding residue in dSlo1A is Thr-337. Ile is hydrophobic residue, but Ala and Thr are hydrophilic ones. We mutated the Ile-323 of wide type mSlo1 into Thr, found that I323T induced the flickery single channel current similar to dSlo1A. This mechanism may help us to understand the behavior of dSlo single channel current.(4) From the computer simulation of the interaction structure between the mSlo1 pore andβ2 N terminal, we can obtain part details of interaction between the two proteins.