| 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 channelswere 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 neuronscell 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.
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