The Role Of Ca²⁺ -activated K⁺ Channel?BK Channel? In Neuropathic Pain

?Background?Pain is one of the most common clinical symptoms.It is generally believed that pain sensation is a survival mechanism and can be used as a warning signal for ongoing or impending tissue damage.However,chronic pain has exceeded its role as an early warning,has become long-lasting and devastating,and is now considered a clinical condition.At present,the number of people suffering from pain has exceeded the sum of the number of heart disease,cancer and diabetes,among which,neuropathic pain is the main cause of chronic pain.The pathogenesis of neuropathic pain is complex and there are no specific and effective therapeutic drugs in clinical practice.Therefore,studying the mechanism of its generation deeply will play an important role in the development of new therapeutic drugs and improve clinical treatment.BK channels are widely expressed in a variety of cells and are distributed in epithelial cells,smooth muscle,skeletal muscle,sensory neurons and so on.Therefore,BK channels participate in many physiological processes,including the regulation of gland secretion,neuronal excitation,neurotransmitter release,etc.Besides,BK channels also involved in the pathological process of various diseases such as neuropathic pain.BK channel is a large-conductance Ca²⁺-activated K⁺channel and plays a role in cell repolarization and cell excitability.When the channel is open,the outflow of potassium ions is manifested as hyperpolarization of the membrane potential;conversely,the decrease in channel activity or the closure of the channel leads to a reduction in the outflow of potassium ions,and the membrane potential appears as depolarization.Therefore,BK channels expressed in neurons may play an important role in the process of transforming acute post-traumatic pain into chronic neuropathic pain by controlling the excitability of neurons and regulating the release of neurotransmitters.In-depth study of gene expression changes of BK channels and the functional changes of channel proteins during the development of neuropathic pain will not only help elucidate the mechanism of development of neuropathic pain but also provide novel and efficient solutions to block the transformation from acute pain to chronic pain.This study intends to investigate the role of BK channels in the pathogenesis of neuropathic pain through behavior test,single cell sequencing techniques and electrophysiological techniques.?Methods?L5-L6 spinal nerve ligation?SNL?were used in Wistar rats to prepare animal models of neuropathic pain.Von Frey fiber was used to measure the threshold of mechanical pain.We take out spinal nerves at different time points after SNL operation and got individual neurons of dorsal root ganglion?DRG?by enzymatic digestion.A single cell sequencing library was constructed by picking single DRG cells into a well-configured lysate using a micropipette,and then the changes of gene expression during the progress of neuropathic pain were analyzed.Since ion channels play an important role in the formation and conduction of nociceptive sensations,the expression of genes encoding the ion channels are further identified.Data analysis reveals a significant change in the expression of BK channel genes during the progression of neuropathic pain after surgery.We further use in vitro single-cell electrophysiological recordings to detect changes in BK channel currents at different time points,verifying its involvement.In this study,we demonstrate that BK channel is involved in the development of neuropathic pain by combining behavior test,single cell sequencing and electrophysiological experiments.?Results??1?The mechanical pain threshold was significantly decreased after the 1st day of SNL operation,and it was basically stable on 3rd-5th day and remained to 21st day or even longer.?2?After isolating the single cells,we found the cells in the DRG can be divided into three major groups depending on the diameter,which are small cells?d<25um??middle cells?25um<d<35um?and large cells?d>35um?.This classification is the precondition of picking different cell types for the following single cell sequencing experiment.?3?The results of single cell sequencing showed that the expression level of BK channel's?subunit?kcnma1?in the DRG small cells was significantly decreased on the3rd day after operation;the gene encoding the?2 subunit?kcnmb2?was apparently decreased on the 1st day after surgery and kept at a low level to the 3rd day after surgery;the expression level of the?4 subunit gene?kcnmb4?increased on the 3rd day and remained elevating to the 5th day.The expression of kcnma1 in DRG medium-sized cells was significantly decreased on the 3rd day after surgery,and further decreased to the lowest level on the 5th day.The?2 gene?kcnmb2?was also significantly decreased on the1st day after operation and kept at a low level to the 3rd day;the?4 subunit gene?kcnmb4?was increased on the 3rd day after operation and increased to the maximum on the 5th day.The results of single cell sequencing show that the components of BK channel changed after surgery and this changes are consistency with mechanical sensitivity,suggesting that BK channel participates in the development of neuropathic pain.?4?Whether changes in gene expression affect the behavior ultimately still requires the detection of channel's function.Therefore,we used electrophysiological methods to analyze the current conditions of the BK channel.We found the characteristics and expression patterns of BK channel current changed in small and medium DRG neurons at different time point.On the 3rd day,the peak amplitude and steady-state of BK channel was significantly reduced.This result indicates that the?subunit of BK channel has changed during the progression of neuropathic pain.By analyzing the time constant of decay,we found that the inactivation of BK current was slowed down after surgery,and the time constants of the slow and fast phases of inactivation of channel currents were increased in both small and middle cells,indicating that?subunits has changed during pain progression.These changes in?and?subunits can lead to a decrease in intracellular potassium efflux and thus to a degree of depolarization,which may lead to hyperalgesia in neuropathic pain.?Conclusion??1?The results of single cell sequencing show that the gene expression level on the 3rd day after nerve injury changed significantly,which indicate the 3rd maybe the key time point for the transition from acute pain to chronic pain.?2?The expression of BK channel genes changed during the progression of neuropathic pain:the expression of?subunits decreased,?2 regulatory subunits decreased,while?4 regulatory subunits increased.?3?The current morphology of the BK channel changed after nerve injury,which is consistent with the change of BK channel genes.These changes can lead to an increased concentration of intracellular potassium ion,which may increase the excitability of the DRG neurons and manifest neuropathic pain symptoms further.