Homomeric and heteromeric ion channels formed from the kainate-type subunits GluR6 and KA2 have very small, but different, unitary conductances
Homomeric and heteromeric ion channels formed from the kainate-type subunits GluR6 and KA2 have very small, but different, unitary conductances
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DOI：
10.1152/jn.1996.76.1.510
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发表时间：
1996-07-01
影响因子：
2.5
通讯作者：
Howe, JR
中科院分区：
文献类型：
--
作者：
Howe, JR
1.用膜片钳技术研究了编码海人藻酸(KA)型谷氨酸受体(GluR)亚单位的重组谷氨酸受体(GluR)离子通道在人胚胎肾细胞(HEK 293)中的表达。用完全编辑的(R)版本的GluR6瞬时或稳定地转染细胞，或与GluR6(R)和KA2.2共转染细胞。获得了KA和谷氨酸激活同质GluR6(R)通道的浓度-反应数据，并用Hill方程拟合出激动剂半最大激活时的浓度(EC(50))、Hill系数(n(H))和最大电流(i-max)。用KA分析7个电池的结果，EC(50)和n(H)的平均值分别为0.47微米和1.47。谷氨酸的对应值分别为32µM和1.21(n=5)。在同一细胞中，KA和谷氨酸的I-max值相似，表明KA和谷氨酸都是同质GluR6(R)通道的完全激动剂。对KA和谷氨酸在表达GluR6(R)的细胞中或在这些细胞的外向斑块中诱发的电流噪声进行频谱密度分析，以获得对同质GluR6(R)通道的表观么正电导(伽马(噪声))的估计。用10微米MKA在15个单元中获得的平均伽马(噪声)值为231fS。外贴噪声分析的对应值为259 FS(n=4)。对六个细胞中谷氨酸诱发的噪声进行频谱密度分析，得出平均伽马(噪声)值为264 fS.4。对GluR6(R)/Ka2表达细胞记录的全细胞电流进行噪声分析，平均伽马(噪声)值为572fS(n=9)。与同构体GluR6(R)通道不同，GluR6(R)/Ka2异构体可被alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic酸(0.2 5~5 mM)激活。GluR6(R)/Ka2通道KA激活的平均EC(50)为1.62uM(n=6)。结果表明，均相GluR6(R)和异构体GluR6(R)/Ka2通道在Femtosiman范围内均具有单一电导。Ka2与GluR6(R)的共同组装导致通道对AMPA和KA具有不同的亲和力，并使单位电导增加两到三倍。
1. Patch-clamp methods were used to study recombinant glutamate receptor (GluR) ion channels expressed in human embryonic kidney cells (HEK 293) after transfection of the cells with cDNAs encoding kainate (KA)-type GluR subunits. Cells were transiently or stably transfected with the fully edited (R) version of GluR6 or they were cotransfected with GluR6(R) and KA2.2. Concentration-response data were obtained for KA and glutamate activation of homomeric GluR6(R) channels and fitted with Hill-type equations to give values for the agonist concentration at half-maximal activation (EC(50)), the Hill coefficient (n(H)), and the maximum current (I-max). Analysis of results obtained in seven cells with KA gave mean values of 0.47 mu M and 1.47 for the EC(50) and n(H), respectively. The corresponding values for glutamate were 32 mu M and 1.21 (n = 5). The I-max values obtained for KA and glutamate in the same cells were similar, suggesting that both KA and glutamate are full agonists at homomeric GluR6(R) channels.3. Spectral density analysis of current noise evoked by KA and glutamate in GluR6(R)-expressing cells, or in outside-out patches from these cells, was used to obtain estimates of the apparent unitary conductance (gamma(noise)) of homomeric GluR6(R) channels. Results obtained with 10 mu M KA in 15 cells gave a mean gamma(noise) value of 231 fS. The corresponding value from analysis of noise in outside-out patches was 259 fS (n = 4). Spectral density analysis of glutamate-evoked noise in six cells gave a mean gamma(noise) value of 264 fS.4. Noise analysis of whole cell currents recorded in GluR6(R)/KA2-expressing cells gave a mean gamma(noise) value of 572 fS (n = 9). Unlike homomeric GluR6(R) channels, GluR6(R)/KA2 heteromers were activated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) (0.25-5 mM). The mean EC(50) for KA activation of GluR6(R)/KA2 channels was 1.62 mu M (n = 6).5. The results indicate that both homomeric GluR6(R) and heteromeric GluR6(R)/KA2 channels have a unitary conductance in the femtosiemen range. The coassembly of KA2 with GluR6(R) results in channels that have a different affinity for AMPA and KA and a two- to threefold larger unitary conductance.