Immunosuppressant Fk506 Of Csa Study, The Role Of Rat Hippocampal Pyramidal Neurons Voltage - Gated Potassium Channels

Background and purpose:Stroke is the third leading cause of death in mankind.About 75%of strokes result from cerebral infarction.It has been demonstrated that hypoxia/ischemic insults to brain cause not only immediate neuronal death(necrosis) within the core of cerebral infarct, but also subsequent delayed neuronal apoptosis in the peri-infarct zone or ischemic penumbra.The delayed neuronal death occurs over days and months after the ischemic insult,which provides a favorable time window for new drugs that are supposed to target the events in the pathophysiological cascades,thus protect brain neurons against ischemic injury.Extensive researches have shown that in Caspase-dependent apoptosis with DNA fragment production,the K⁺ outward influx increases and intracellular K⁺ concentration decreases in the early process of apoptosis.Among so many different kinds of potassium channels,the delayed rectifier K+ channel(I_K) has been proposed to play the most important role in enhancement of potassium outward current during the early part of apoptosis due to its high conductance and none or slow inactivation.A series of in vitro studies have shown that enhanced delayed rectifier K⁺ channel induces apoptosis of brain neurons,while blocking K⁺ outflow through the K+ channel promotes neuronal survival.Immunosuppressant drugs tacrolimus(FK506) and cyclosporin A (CsA) are the specific inhibitiors of calcineurin.It has been demonstrated that FK506 and CsA,have shown neuroprotective action in defferent models of brain ischemia.However,it is unclear whether the neuroprotective action results from blocking the K⁺ channel.The aim of our study is to investigate the effects of FK506 and CsA on the voltage-activated K⁺ currents in acutely dissociated CA1 pyramidal neurons of rat hippocampus,and to explore whether the effects is mediated through inhibition of calcineurin.Methods:Whole-cell current-clamp recording was made in CA1 pyramdal neurons in rat hippocampal slices;Whole-cell voltage-clamp recording was made in dissociated hippocampal CA1 pyramdal neurons of rat.Results:The research includes tow parts,the results as following:Part1:Whole-cell current-clamp recording was made in CA1 pyramidal neurons in rat hippocampal slices,FK506(50μM) and CsA(100μM) did not significantly alter the passive electrical properties of hippocampal CA1 pyramidal neurons,but slowed down the repolarizing phase of the action potential.Part 2:Whole-cell voltage-clamp recording was made in acutely dissociated CA1 pyramidal neurons of rat hippocampus,superfusion of FK506(0.01-100μM) selectively inhibited the delayed rectifier K+ current(I_K) in a concentration-dependent manner,with an IC50 value of 13.2±4.9μM,but did not affect the fast transient K+current(I_A).The inhibition of I_K by FK506(10μM) had a rapid onset,and then gradually reached a steady-state level.The inhibition was voltage-dependent, became more potent when the currents were elicited by strong depolarization.Moreover,FK506(10μM) caused marked negative shifts of the steady-state activation and inactivation curves of I_K,and accelerated its recovery from inactivation.We also found:(1) Intracellular dialysis of FK506(30μM) was ineffective;(2) The inhibition of I_K by FK506(10μM) persisted under the low-Ca²⁺ conditions that blocked the basal activity of protein phosphatase 2B (calcineurin);(3) Rapamycin did not antagonize FK506 but mimicked it. These suggesting that the inhibition of the I_K was not caused by the complex of FK506/FKBP-12,or the subsequent inhibition of calcineurin.In acutely dissociated CA1 pyramidal neurons of rat hippocampus, CsA(10-100μM) selectively inhibited the delayed rectifier K⁺ current (I_K) in a concentration-dependent manner,but did not affect the fast transient K⁺ current(I_A).The inhibition of I_K by CsA(100μM) had a rapid onset,and immediately reached a level of nearly 80%of the maximum inhibition.The inhibition was voltage-independent.CsA did not affect the kinetic properties of I_K.Intracellular dialysis of CsA(100μM) had no effect on I_K;the inhibition of I_K by CsA(100μM) persisted under the low Ca²⁺ conditions that blocked the basal activity of calcineurin;suggesting that the inhibition of the I_K was not caused by the complex of CsA/Cyc,or the subsequent inhibition of calcineurin. Conclusions:the present study demonstrates that FK506 and CsA selectively inhibited the delayed rectifier K⁺ current(I_K),and they may exert a direct inhibition on the delayed rectifier K⁺ channel without involvement of calcineurin.It provides theorical foundation for further studing the neuroprotective action of immunosuppressant drugs and seeking for more effective neuroprotective drugs.