To Probe The Interaction Sites Between The MSlo1 Pore And The NH2 Terminus Of The Hβ2 Subunit, And Study On The Mechanism Of Desensitization Of Pain Receptors TRPV1

The developments of modern life science and technology have greatly influenced our understanding in the evolution of human lives.The studies on ion channels are not only related to the signal occurrence and transmission,but also involved in the molecular mechanisms of hereditary or inhereditary diseases.To explore the relationships between the structure and function of the ion channels and study on the mechanisms of gating and inactivation of ion channels are very important,and also will benefit the society and human health.There are two parts in this study.The first part is to study on the interaction ofβ2 subunit and the pore forming by mslo 1 subunit of Calcium- and voltage- activated large conductace potassium channel(Maxik channel,also termed BK channel),the other one is focus on the molecular mechanisms of desensitization of pain receptors TRPV1 channel.BK channel extensively exists in excitable cells,especially in neural system,which plays a significant physiological role in mediating the concentration of intracellular calcium ions and the membrane potential.To date,the voltage dependent potassium channel(Kv channel) has been deeply studied.BK channel shares structural and functional similarities with Kv channel,i.e.,they both can combine auxiliaryβsubunits, and have a typical N-type inactivation induced by the N terminals ofβsubunits.However there are also some differences between them.Unlike Kv channels,the pore blockers (such as TEA and QX-314) of K channels can not slow down the inactivation rate of BK channels.Why are they so different in this issue? Do the N terminals ofβsubunits indeed enter into the pore forming by mslol? Where or what are the interaction sites between them during inactivation? To address these questions,we presented this study here.When mutating the first three amino acids of hβ2 N terminal FIW into FWI,we surprisingly found that the property of recovery curve was changed into bi-exponent from mono-exponent,but the FWI mutant has no effects on the inactivation process of the BK channels.Using this behavior of recovery of FWI,we tried to elucidate the relationship between them during inactivation by mutating the hydrophobic amino acids within the BK pore to less hydrophobic one,alanine.At last,we found that Ile 323 is the main interaction site betweenαsubunit andβ2 subunit during the process of inactivation.Our data also support that:(1) Among all the S6 mutants,mSlol-I323A coexpressed with hβ2-FWI,the slow component of the recovery was maximally reduced.That is,its recovery curve can be almost fitted with a mono-exponential function.It indicates that I323 is the main interaction site withβ2 subunit during the inactivation.In addition,the fast components of M314A and V319A were extrodinarily faster,sugesting that the two points have certain effects during the process of inactivation.Based on the linear structure relationship of FWI, we inferred the following interaction relationships:I323-I,V319-W,M314-F,and I323 played the leading role.(2) Despite the sensitivity of V319A to QX-314 is less higher than others,there is no specific binding site for QX-314 in the pore.A one step non-competition model can well explain our data other than a two-step model.The reason is that Ile-323 is the last residue in the pore,the interaction sites ofβ2 inactivation domain are located on the outer entrance of the pore,the interaction sites of QX-314 are inside of the pore,and therefore, QX-314 might not impact the process of inactivation.This non-competitive model explained well that why intracellular blockers do not slow down the inactivation process of BK channels.These data can help us understanding the structure and the gating mechanisms of BK channels.(3) Another intriguing phenomenon was seen with the mutant I323A.In comparison to the wide-type BK channels,the mutation I323A showed an obiviously outward rectification in both single channel recordings and macro patches.And also the single channel current of I323A is much flickery,even comparing to that of the single channel current of dSlo1.These data support that the residue Ile-323 also mediates the gating of BK channel.The corresponding residue in dSlo1 is Thr-337.Ile is a higher hydrophobic residue in comparison to Ala and Thr.In the following studies,we found that mutation I323T induced a similarly flickery single channel current similar as wild-type dSlo1 channel,which was reported by Guo et al in the Biophys J.(Guo et al,Biophys J. 2008 May 1;94(9):3714-25).This mechanism may also help us to understand the behavior of dSlo single channel current.Adaptation is a common feature of many sensory systems.But its occurrence to pain sensation has remained elusive.In the second part of this study,we address the problem at the receptor level and show that the capsaicin ion channel TRPV1,which mediates nociception at the peripheral nerve terminals,processes properties essential to the adaptation of sensory responses.Ca²⁺ influx following the channel opening caused a profound shift(～14 folds) of the agonist sensitivity,but did not alter the maximum attainable current.The main points we got are as following:(1) Ca²⁺ influx following the channel opening caused a profound shift of the agonist sensitivity,about 14 folds,but did not alter the maximum attainable current.The shift was adequate to render the channel irresponsive to normally saturating concentrations,leaving the notion that the channel became no longer functional after desensitization.(2) By simultaneous patch-clamp recordings and total internal reflection fluorescence(TIRF) imaging,it was shown that the depletion of phosphatidylinositol 4,5-bisphosphate(PIP2) induced by Ca²⁺ influx had a rapid time course synchronous to the desensitization of the current.The extent of the depletion was comparable to that by rapamycin-induced activation of a PIP2 5-phosphatase,which also caused a significant reduction of the agonist sensitivity without affecting maximum response.The change induced by rapamycin accounted for～57%of the overall shift resulting from the desensitization by Ca²⁺ influx,indicating that the depletion of PIP2 constitutes a prominent component of the adaptation of the channel.(3) We showed that the adaptation of the channel appeared to be stimulus-dependent. It appeared that the desensitized channel could only be reactivated by a stimulus higher than used for desensitization.We do not know how the channel "remember" the desensitizing conditions,and hypothesize the causal is the transient profile of the submembrane[Ca²⁺]could differ with different stimuli.The dynamic of Ca²⁺ waves are known to affect a variety of cellular processes.Conceivably,it may also contribute to the regulation of ion channels.