Brock M W, Lebaric Z N, Neumeister H, DeTomaso A, Gilly W F
Neurosciences Program of Stanford University, CA 94305, USA.
J Membr Biol. 2001 Mar 15;180(2):147-61. doi: 10.1007/s002320010066.
SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) alpha-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (IK) if cultured at the standard postinfection temperature of 27 degrees C. At 18 degrees C, less SqKv1A protein is produced than at 27 degrees C, but cells with IK currents are much more numerous and can survive for at least 20 days postinfection (vs. approximately 5 days at 27 degrees C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (approximately 3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of IK inactivation in 20 mM external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL IK is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.
SqKv1A是一种编码Kv1（震颤型）α亚基的cDNA，仅在鱿鱼星状神经节的巨大轴突和亲代巨大纤维叶（GFL）神经元中表达。我们将SqKv1A整合到重组杆状病毒中，以便在昆虫Sf9细胞系中表达。全细胞膜片钳记录显示，如果在27摄氏度的标准感染后温度下培养，很少有细胞显示出功能性钾电流（IK）。在18摄氏度时，产生的SqKv1A蛋白比在27摄氏度时少，但具有IK电流的细胞数量要多得多，并且在感染后至少可以存活20天（相比之下，在27摄氏度时约为5天）。SqKv1A在Sf9细胞中的激活和失活动力学比GFL神经元中的天然通道慢（分别约为3倍和10倍），但具有相似的电压依赖性。这两种细胞类型在电导和失活的稳态电压依赖性方面仅表现出细微差异。在20 mM外部钾离子存在下，两种细胞类型的IK失活速率相同，但失活对外部四乙铵（TEA）和钾离子的敏感性不同：Sf9细胞中SqKv1A的失活被外部TEA和钾离子减慢，而GFL IK的失活在很大程度上不敏感。根据可能特定于细胞类型的因素讨论了功能差异，包括GFL神经元中目前尚未鉴定的Kv1亚基的存在，这些亚基可能与SqKv1A形成异源多聚体。
