Martel J, Dupuis G, Deschênes P, Payet M D
Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
J Membr Biol. 1998 Jan 15;161(2):183-96. doi: 10.1007/s002329900325.
cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (delta 459-523) form that lacks the putative PKA Ser468 phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some straining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K+ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of -80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to -40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were -5.7 and -9.9 mV (slope factor) and -35 mV (half activation, V0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and -17.3 and -39.0 mV (V0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from -30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at -30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mM KCl was 1.3 +/- 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni2+ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 +/- 7.3% and 38.7 +/- 9.4% inhibition of the full-length and the truncated channels, respectively, 8-BrcAMP induced a 39.4 +/- 5.4% inhibition of the full-length channel but had no effect (8.6 +/- 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kv beta 1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kv beta 2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kv beta-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced.
编码全长hKv1.3淋巴细胞通道和缺少假定的蛋白激酶A（PKA）丝氨酸468磷酸化位点的C末端截短形式（δ459 - 523）的互补DNA（cDNA）被稳定转染到人胚肾（HEK）293细胞中。对转染细胞的免疫染色显示，在全长通道的情况下，质膜上的分布是均匀的，而在截短通道的情况下则观察到聚集现象。在这两种情况下，在细胞质中均发现了一些染色，表明存在活跃的生物合成过程。采用膜片钳技术在全细胞模式下对钾离子电流进行分析，结果显示，从 - 80 mV的保持电位（HP）去极化至40 mV会引发2至10 nA的外向电流。电流阈值为正至 - 40 mV，且电流幅度以电压依赖性方式增加。全长通道和截短通道的激活参数分别为 - 5.7和 - 9.9 mV（斜率因子）以及 - 35 mV（半激活，V0.5）。全长通道和截短通道的失活特征分别为14.2和24.6 mV（斜率因子）以及 - 17.3和 - 39.0 mV（V0.5）。全长通道在 - 30至40 mV电位范围内的激活时间常数从18毫秒降至12毫秒，而失活时间常数从 - 30 mV时的6600毫秒降至40 mV时的1800毫秒。在140 mM氯化钾溶液中孵育的细胞中，在40 mV时测得的单位电流幅度为1.3±0.1 pA，单位电导为34.5 pS，零电流电压为0 mV。两种形式的通道均受到四乙铵（TEA）、4 - 氨基吡啶（4 - AP）、镍离子（Ni2 +）和蝎毒素的抑制。与天然（Jurkat）淋巴细胞Kv1.3通道被PKA和蛋白激酶C（PKC）完全抑制不同，添加佛波酯（TPA）分别导致全长通道和截短通道受到34.6±7.3%和38.7±9.4%的抑制，8 - 溴腺苷 - 3',5' - 环一磷酸（8 - BrcAMP）诱导全长通道受到39.4±5.4%的抑制，但对截短通道无影响（8.6±8.3%）。用碱性磷酸酶进行细胞透析没有效果，这表明转染通道对PKA和PKC敏感性降低并非由于通道已被磷酸化。膜片提取物实验表明，hKv1.3通道对PKA和PKC部分敏感。共转染Kvβ1.2亚基导致全长通道失活时间常数的值降低，但未改变其对PKA和PKC的敏感性。共转染的Kvβ2亚基没有影响。我们的结果表明，hKv1.3淋巴细胞通道在转染到Kvβ阴性的HEK 293细胞系中时保留了其电生理特性，但其对PKA和PKC调节的敏感性显著降低。
