Molecular Mechanisms Of Calcium-dependent Gating In Calcium-activated Chloride Channels

Calcium-activated chloride channel （CaCCs） is a chloride channel involved in multiplephysiological functions, such as, in chloride ion secretion of the epithelial cells, olfactorytransduction, and smooth muscle contraction. It is very difficult to separate CaCCs mediatedcurrent from calcium-dependent the cation current and non-calcium-dependent chloride ion, sothe study of their electrophysiological properties lagged far behind that of other ion channels.Recently, TMEM16A is recognized as the molecular basis of CaCCs, which provides greatpossibility for further study of the electrophysiological characteristics.Calcium and voltage dual-dependent activation mechanism of the TMEM16A channel isseemed as typical electrophysiological properties. However, there does not exist known calciumbinding domain in the TMEM16A channel internal, such as, CaM’s EF-hand and BK channel’s―Ca²⁺-bowl‖, etc. So it is vital to determine the TMEM16A channel calcium ion binding site instudying the premise calcium-dependent activation mechanisms.In this paper, in combination with the study of Pang Chunli’s theoretical simulation and GaoChongsen’s experiment results, who are our group members, we studied the activationmechanism of TMEM16A from to aspects:1、 Study on the Ca²⁺binding sites of TMEM16ABy combination of molecular dynamics theory method with site-directed mutagenesisexperiment and patch-clamp technique, TMEM16A channel’s Ca²⁺-binding site is studtied andsome results were obtained:（1） In the first Loop of TMEM16A channel, carboxyl oxygen atoms of the D439, E444andE447, and oxygen atoms of the adjacent three water molecules can bind directly to thecalcium ion, form a possible Ca²⁺-binding site.（2） In the formation of the calcium binding site, D439and E444each contributes one oxygenatom, and the E447contributes two oxygen atoms in binding calcium ion, with in which the E447for channel affinity has the strongest impact on calcium ion.（3） In the first Loop of TMEM16A channel, the D452and E457do not affect channel gatingkinetics behavior in the patch-clamp results, although they can also form a calcium ionbinding site in the molecular dynamic simulations.2、Preliminary study of the divalent cations in the TMEM16A channel activation progressOur group and other laboratories’s studies show that different divalent cations can activateTMEM16A as well. Examined action of the calcium-dependent activation mechanism fordifferent divalent cation activation is very important in TMEM16A’s gating mechanism. Till now,research on the relationship of the divalent ions and calcium ions in the activation TMEM16Achannel process has not yet been reported.In this paper, using inside-out patch clamp recording mode, we studied of Sr²⁺, Ni²⁺andCa²⁺interact with TMEM16A channel adopted in transient-transfected HEK293cells. Bystudying the effect of different divalent cations on CaCCs, thereby judging whether there issynergy between the divalent cations. The experimental results showed that:（1） When the co-existence of the two divalent cations (Ca²⁺+Sr²⁺, Ca²⁺+Ni²⁺), can not be agreater degree of activation of TMEM16A channel.（2） When Sr²⁺or Ni²⁺in non-saturation concentration, Ca²⁺compensatory increase TMEM16Achannel currents.According to the above results, we speculated that Sr²⁺, Ni²⁺or Ca²⁺is likely through thesame mechanism to activate TMEM16A.