下丘脑神经元BK_（Ca）通道特性及温度依赖性
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摘要
中枢性体温调节的本质和调控因素，是高温环境医学和发热病理学研究领域的热点。下丘脑视前区和下丘脑前区(POAH)为恒温动物的体温调节中枢。然而该部位神经元参与体温调节反应的温度敏感机制仍有许多未知数，更缺乏作为神经生物电信号基础的离子通道在这一环节中如何发挥作用的报道。本实验研究了SD乳鼠下丘脑神经元大电导钙激活钾通道(Large-conductance Ca~(2+)-activated potassium channels，BK_(Ca))特性的温度依赖性及磷酸化对其温度依赖性的影响，以求为体温调节机制的最终阐明提供重要的理论依据。
目的：研究新生SD乳鼠下丘脑神经元钙激活钾通道电生理特性的温度依赖性及其调控因素，以说明BK_(Ca)通道在机体温度感受与体温调节中发挥的重要作用。
方法：急性分离＊D乳鼠下丘脑神经元，采川腴片钳技术的内面问
外式记录大电导钙激活钾通道活动。
结果：门）当细胞内钙离子浓度OC／勺i）为 0刀引。id几时厂始出
现＊几。通道的活动，且通道的开放概率＊耶11h时劝ilityP。）随着＊／”］；
的增加而增加。当＊］／［K勺J140川 时，通道电导为17lps。随着温
度的增加，BKc。通道的电导及开放概率显著增加，并可出现二级或多级
开放。29℃、33℃、37℃时电导的o。值为 1．ZI、1．83及 2．58，开放概
率的o。为2刀5、4．42和3．34。通道的开放时间、关闭时间均表现为温
度依赖性缩短，尤以 T卜 T。。表现显著。Q）浴槽液中加入ATI叫使
BKC。通道活动减弱，这种效应可被预先加人蛋白激酶A阻断剂11E9取
消；如果先加 ATP，后加入的 H－89可将 ATP对通道活动的抑制作用翻
转。经磷酸化修饰后的BKC。通道29℃、33℃、37”C时电导的q（）值分
别为 1．18、1j 8及 1．42，较磷酸化前明显减小。通道开放概率丧失了原
有的温度敏感性。这种改变是可逆的，胞浆侧加入P皿阻断剂1｝89可
恢复涸道的温度敏感性。
结论：（）下丘脑神经元 BKk通道是同时具有高度的 C4”敏感性
及电压依赖性的双重门控皿道，且两种门控机制互相调控，这充分证实
了 BKC。通道可将细胞信使 Cah的调节与神经元电兴奋性的调节有机的
偶联起来，进一步确定了其在细胞信号转导中的重要作用。（二）升高温
度增强该通道的活性，降低温度则使其活性减弱。通道动力学研究表叭
其开放概率的增高是通过开放频率的大幅度升高而实现的。8闪。通道的
这种高度的温度敏感性提示其可能是下丘脑体温调节中枢的感温装B，
将温度这个物理信号与神经元的兴奋性有机的联系到一起。门）磷酸化
抑制＊兄。通道的活动，而这种抑制作用可被P以特异性阻断剂1｝89
翻转，表明PKA介导了ATP对通道活动的抑制作用。提示BK。。通道可
同时受蛋白激酶的磷酸化与蛋白磷酯酶的去磷酸化的同时调控，而BKC。
通道在某一时间点的磷酸化状态取决于蛋EI激酶的磷酸化、蛋白磷酯酶
的之磷酸化之间的平衡。N）经磷酸化修饰后，BKC＊ 道的活性及温反
敏肘仕均表现出可逆性降低，捉示磷酸化修饰可能参与了下丘脑BKc。
通道的体温调节功能。
The mechanism of thermoregulation is the hot spot of hypertheimia environmental medicine and febrile pathology. It is well know that the preoptic and anterior hypothalamus (POAII) is the heat regulating center of homeotherm. But little is know about the mechanism of temperature sensitive neurons in hypothalamus, not less the role of ion channels play in temperature regulation. In this experiment, the inside-out configurations of patch-clamp techniques were used to study the physiological properties, phosphorylation modulation and temperature sensitivity of BKca channel in hypothalamic neurons of newborn SD rats.
Objective : Studing the physiological properties. phosphorylation modulation and temperature sensitivity of calcium-activated potassium channels (BKca) in hypothalamic neurons of newborn SD rats in order to elucidate the role of BICca channel in temperature regulation.
Methods: hypothalamic neurons cells were acutely isolated from the brain of SD rats 1-3 days both sexes. The date was recorded at single-channel level using inside-out configuration of patch-clamp techniques.
Results: The most fundamental response that can be measured was the fraction of channels that were open as a function of [Ca2+]j. BKca channels
were activated firstly at 0.05 u mol/L [Ca2+]i with membrane potential of +20mV. The Po (Open Probability) of the channels depended on [Ca2+];. Conductance of single BKCa channel was approximately 171 pS with symmetrical 140mmol/1 K+ on both sides of excised membrane. As the temperature increased, peak conductance and Po of the channel increased greatly and burst opening could be found, when the temperature was at 29C , 33C and 37C, the Q10 of the conductance and Po was 1.21, 1.83, 2.58 and 2.05, 4.42, 3.34 respectively. The temporal analysis of the channels showed that the currents generated an exponential distribution of open time and a biexponential distribution of shut time in all patches. Both open time constant and closed time constant had temperature-dependent reductions with increasing temperature, especially for To and T c2.
The intracellular application of ATP induced a markedly decrease of Po through the increased open frequency without affecting the unitary conductance and voltage dependence of BKca channel. This effect could be inhibited and reversed by the application of H-89 before and after ATP. when the temperature was at 29C , 33C and 37C, the Q10 of the conductance of BKca channels in the presence of ATP without H-89 was 1.18 , 1.18 and 1.42. The temperature sensitivity of Po of BKca channels disappeared completely and opposite results could be found in the absence and presence of H-89.
Conclusion : (1) The calcium-activated potassium channels in hypothalamic neurons had high Ca2+ sensitivity and voltage dependence. These channels coupled the membrane excitability to intracellular Ca2+ concentration and were believed to play an important role in intracellular signaling. (2) Both unitary conductance and Po of BKca channel were markedly sensitive to temperature. Raising temperature enhanced the activities of BKca channels, meanwhile decreased temperature had opposite change. It seems reasonable to assume that BKca channels in hypothalamic neurons is the thermoreceptor of heat regulating ceter which link the neurons
cell excitability and temperature together. (3) In our study, ATP weaken the activities of BKca channels. This effect could be inhibited and reversed by H-89, which indicated the participation of PKA. It is possible that phosphorylation and dephosphorylation modulation of BKca channels interact to play an important role in the functional profile and BKCa channels is in a mixed phosphorylation state. (4) Phosphorylation modulation decreased activitis and temperature sensitivity of BKca channel reversibly, which suggested the participation of protein phosphorylation in the temperature regulation. More researches on it may be helpful for us to elucidate the mechanism of temperature regulation of animals.
目的：研究新生SD乳鼠下丘脑神经元钙激活钾通道电生理特性的温度依赖性及其调控因素，以说明BK_(Ca)通道在机体温度感受与体温调节中发挥的重要作用。
方法：急性分离＊D乳鼠下丘脑神经元，采川腴片钳技术的内面问
外式记录大电导钙激活钾通道活动。
结果：门）当细胞内钙离子浓度OC／勺i）为 0刀引。id几时厂始出
现＊几。通道的活动，且通道的开放概率＊耶11h时劝ilityP。）随着＊／”］；
的增加而增加。当＊］／［K勺J140川 时，通道电导为17lps。随着温
度的增加，BKc。通道的电导及开放概率显著增加，并可出现二级或多级
开放。29℃、33℃、37℃时电导的o。值为 1．ZI、1．83及 2．58，开放概
率的o。为2刀5、4．42和3．34。通道的开放时间、关闭时间均表现为温
度依赖性缩短，尤以 T卜 T。。表现显著。Q）浴槽液中加入ATI叫使
BKC。通道活动减弱，这种效应可被预先加人蛋白激酶A阻断剂11E9取
消；如果先加 ATP，后加入的 H－89可将 ATP对通道活动的抑制作用翻
转。经磷酸化修饰后的BKC。通道29℃、33℃、37”C时电导的q（）值分
别为 1．18、1j 8及 1．42，较磷酸化前明显减小。通道开放概率丧失了原
有的温度敏感性。这种改变是可逆的，胞浆侧加入P皿阻断剂1｝89可
恢复涸道的温度敏感性。
结论：（）下丘脑神经元 BKk通道是同时具有高度的 C4”敏感性
及电压依赖性的双重门控皿道，且两种门控机制互相调控，这充分证实
了 BKC。通道可将细胞信使 Cah的调节与神经元电兴奋性的调节有机的
偶联起来，进一步确定了其在细胞信号转导中的重要作用。（二）升高温
度增强该通道的活性，降低温度则使其活性减弱。通道动力学研究表叭
其开放概率的增高是通过开放频率的大幅度升高而实现的。8闪。通道的
这种高度的温度敏感性提示其可能是下丘脑体温调节中枢的感温装B，
将温度这个物理信号与神经元的兴奋性有机的联系到一起。门）磷酸化
抑制＊兄。通道的活动，而这种抑制作用可被P以特异性阻断剂1｝89
翻转，表明PKA介导了ATP对通道活动的抑制作用。提示BK。。通道可
同时受蛋白激酶的磷酸化与蛋白磷酯酶的去磷酸化的同时调控，而BKC。
通道在某一时间点的磷酸化状态取决于蛋EI激酶的磷酸化、蛋白磷酯酶
的之磷酸化之间的平衡。N）经磷酸化修饰后，BKC＊ 道的活性及温反
敏肘仕均表现出可逆性降低，捉示磷酸化修饰可能参与了下丘脑BKc。
通道的体温调节功能。
The mechanism of thermoregulation is the hot spot of hypertheimia environmental medicine and febrile pathology. It is well know that the preoptic and anterior hypothalamus (POAII) is the heat regulating center of homeotherm. But little is know about the mechanism of temperature sensitive neurons in hypothalamus, not less the role of ion channels play in temperature regulation. In this experiment, the inside-out configurations of patch-clamp techniques were used to study the physiological properties, phosphorylation modulation and temperature sensitivity of BKca channel in hypothalamic neurons of newborn SD rats.
Objective : Studing the physiological properties. phosphorylation modulation and temperature sensitivity of calcium-activated potassium channels (BKca) in hypothalamic neurons of newborn SD rats in order to elucidate the role of BICca channel in temperature regulation.
Methods: hypothalamic neurons cells were acutely isolated from the brain of SD rats 1-3 days both sexes. The date was recorded at single-channel level using inside-out configuration of patch-clamp techniques.
Results: The most fundamental response that can be measured was the fraction of channels that were open as a function of [Ca2+]j. BKca channels
were activated firstly at 0.05 u mol/L [Ca2+]i with membrane potential of +20mV. The Po (Open Probability) of the channels depended on [Ca2+];. Conductance of single BKCa channel was approximately 171 pS with symmetrical 140mmol/1 K+ on both sides of excised membrane. As the temperature increased, peak conductance and Po of the channel increased greatly and burst opening could be found, when the temperature was at 29C , 33C and 37C, the Q10 of the conductance and Po was 1.21, 1.83, 2.58 and 2.05, 4.42, 3.34 respectively. The temporal analysis of the channels showed that the currents generated an exponential distribution of open time and a biexponential distribution of shut time in all patches. Both open time constant and closed time constant had temperature-dependent reductions with increasing temperature, especially for To and T c2.
The intracellular application of ATP induced a markedly decrease of Po through the increased open frequency without affecting the unitary conductance and voltage dependence of BKca channel. This effect could be inhibited and reversed by the application of H-89 before and after ATP. when the temperature was at 29C , 33C and 37C, the Q10 of the conductance of BKca channels in the presence of ATP without H-89 was 1.18 , 1.18 and 1.42. The temperature sensitivity of Po of BKca channels disappeared completely and opposite results could be found in the absence and presence of H-89.
Conclusion : (1) The calcium-activated potassium channels in hypothalamic neurons had high Ca2+ sensitivity and voltage dependence. These channels coupled the membrane excitability to intracellular Ca2+ concentration and were believed to play an important role in intracellular signaling. (2) Both unitary conductance and Po of BKca channel were markedly sensitive to temperature. Raising temperature enhanced the activities of BKca channels, meanwhile decreased temperature had opposite change. It seems reasonable to assume that BKca channels in hypothalamic neurons is the thermoreceptor of heat regulating ceter which link the neurons
cell excitability and temperature together. (3) In our study, ATP weaken the activities of BKca channels. This effect could be inhibited and reversed by H-89, which indicated the participation of PKA. It is possible that phosphorylation and dephosphorylation modulation of BKca channels interact to play an important role in the functional profile and BKCa channels is in a mixed phosphorylation state. (4) Phosphorylation modulation decreased activitis and temperature sensitivity of BKca channel reversibly, which suggested the participation of protein phosphorylation in the temperature regulation. More researches on it may be helpful for us to elucidate the mechanism of temperature regulation of animals.
引文
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