The Mechanisms Of BK Channel Interacts With TRPV1 Channel And Ca_v3.2 Channel

Large-conductance Ca²⁺ -activated K⁺ channels?BK channels?can response to changes in membrane potentials and elevation of cytosolic calcium ions(Ca²⁺)and regulates many physiological processes,including action potential firing,neurotransmitter release,hormone secretion and muscle contraction.In many tissues,BK channels are not only regulated by their auxiliary subunits,they can also resemble with other proteins into protein complex,especially calcium channels,which is the structural basis of their diverse functions.Here we show that BK channels can functionaly coupled with TRPV1 channels in rat Dosal Root Ganglion?DRG?cells and Ca_v3.2 channels in brain.We have deeply studied their functional properties by molecular cloning,co-immunoprecipitation,immunofluorescence and electrophysiological techniques.And we also tried to locate the single molecule of channel proteins and reveal their spatial distribution by Photoactivated localization microscopy?PALM?.The transient receptor potential vanilloid receptor 1?TRPV1?channel is a nonselective cation channel activated by a variety of exogenous and endogenous physical and chemical stimuli,such as temperature??>42??,capsaicin,a pungent compound in hot chili peppers,and allyl isothiocyanate.Both BK channels and TRPV1 channels exist in DRG cells.However,it is unknown whether the TRPV1 channels are coupled with the BK channels.Using patch-clamp recording combined with an infrared laser device,we found that BK channels could be activated at 0 mV by a Ca²⁺ influx through TRPV1 channels not the intracellular calcium stores in submilliseconds.The local calcium concentration around BK is estimated over 10?M.The crosstalk could be affected by 10 mM BAPTA,whereas 5 mM EGTA was ineffectual.Fluorescence and co-immunoprecipitation experiments also showed that BK and TRPV1 were able to form a TRPV1-BK complex.Furthermore,we demonstrated that the TRPV1-BK coupling also occurs in DRG cells,which plays a critical physiological role in regulating the "pain" signal transduction pathway in the peripheral nervous system.Ca_v3.2 channel is one of the low-voltage activated calcium channels,of which can be activated at hyperpolarized potentials?-70 to-60 mV?.From our electrophysiological experiments,we found that BK currents were activated by calcium influx produced by Ca_v3.2 channels,of which the maximum current was produced by a 30 ms conditioning step of-30 mV.Co-immunoprecipitation experiments also showed that BK and Ca_v3.2 were able to form a Ca_v3.2-BK complex.However,this protein complex has no effect on the kinetics characteristics of individuals.The local calcium concentration around BK is estimated about 1?M from the voltage-and calcium-dependence of inactivation time constants of BK channels.To further verify the exact distance between BK channels and its partner proteins,we tried to obtain the single molecule images of channel proteins by PALM microscopy.We found that both BK and Ca_v3.2 channel assembled into irregular clusters.Each cluster was assembled with many single molecules but they were not fully co-localized.This result revealed the structural basis of Ca_v3.2-BK complex at the molecule level and provided a model for studying the molecular mechanisms of protein interaction.Also PALM microscopy servers as a new tool in the research of many vital activities,such as gene expression,signaling pathways and protein-protein interaction.