Sokolov Maxim V, Shamotienko Oleg, Dhochartaigh Sorcha Ní, Sack Jon T, Dolly J Oliver
International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland.
Neuropharmacology. 2007 Aug;53(2):272-82. doi: 10.1016/j.neuropharm.2007.05.008. Epub 2007 May 21.
At least five subtypes of voltage-gated (Kv1) channels occur in neurons as tetrameric combinations of different alpha subunits. Their involvement in controlling cell excitability and synaptic transmission make them potential targets for neurotherapeutics. As a prerequisite for this, we established herein how the characteristics of hetero-oligomeric K(+) channels can be influenced by alpha subunit composition. Since the three most prevalent Kv1 subunits in brain are Kv1.2, 1.1 and 1.6, new Kv1.6-1.2 and Kv1.1-1.2 concatenated constructs in pIRES-EGFP were stably expressed in HEK cells and the biophysical plus pharmacological properties of their K(+) currents determined relative to those for the requisite homo-tetramers. These heteromers yielded delayed-rectifier type K(+) currents whose activation, deactivation and inactivation parameters are fairly similar although substituting Kv1.1 with Kv1.6 led to a small negative shift in the conductance-voltage relationship, a direction unexpected from the characteristics of the parental homo-tetramers. Changes resulting from swapping Kv1.6 for Kv1.1 in the concatemers were clearly discerned with two pharmacological agents, as measured by inhibition of the K(+) currents and Rb(+) efflux. alphaDendrotoxin and 4-aminopyridine gave a similar blockade of both hetero-tetramers, as expected. Most important for pharmacological dissection of channel subtypes, dendrotoxin(k) and tetraethylammonium readily distinguished the susceptible Kv1.1-1.2 containing oligomers from the resistant Kv1.6-1.2 channels. Moreover, the discriminating ability of dendrotoxin(k) was further confirmed by its far greater ability to displace (125)I-labelled alphadendrotoxin binding to Kv1.1-1.2 than Kv1.6-1.2 channels. Thus, due to the profiles of these two channel subtypes being found to differ, it seems that only multimers corresponding to those present in the nervous system provide meaningful targets for drug development.
至少有五种电压门控（Kv1）通道亚型以不同α亚基的四聚体组合形式存在于神经元中。它们参与控制细胞兴奋性和突触传递，使其成为神经治疗的潜在靶点。作为前提条件，我们在此确定了α亚基组成如何影响异源寡聚体K(+)通道的特性。由于大脑中三种最普遍的Kv1亚基是Kv1.2、1.1和1.6，因此将新的Kv1.6 - 1.2和Kv1.1 - 1.2串联构建体稳定表达于pIRES - EGFP中，在HEK细胞中表达，并相对于相应的同源四聚体确定其K(+)电流的生物物理和药理学特性。这些异源四聚体产生延迟整流型K(+)电流，其激活、失活和失活参数相当相似，尽管用Kv1.6替代Kv1.1导致电导 - 电压关系出现小的负向偏移，这一方向与亲本同源四聚体的特性预期不符。通过两种药理学试剂测量K(+)电流抑制和Rb(+)外流，清晰地辨别出串联体中用Kv1.6替换Kv1.1所导致的变化。α - 树突毒素和4 - 氨基吡啶对两种异源四聚体产生类似的阻断作用，这是预期的。对于通道亚型的药理学剖析而言，最重要的是，树突毒素（k）和四乙铵能够轻易区分含有敏感的Kv1.1 - 1.2的寡聚体和抗性的Kv1.6 - 1.2通道。此外，树突毒素（k）的区分能力通过其比Kv1.6 - 1.2通道更能有效取代（125）I标记的α - 树突毒素与Kv1.1 - 1.2的结合而得到进一步证实。因此，由于发现这两种通道亚型的特征不同，似乎只有与神经系统中存在的那些相对应多聚体才为药物开发提供有意义的靶点。
