Light-activated ion channels for neural control

人工控制神经元活动的能力无论在实验上对于理解神经回路，还是在治疗上对于补偿神经结构的损伤或退化都是重要的。通常使用的神经控制的电气或化学方法是侵入性的，并且可能在空间和时间上不准确。基因的靶向表达 编码K+或C1-通道已被用来使特定神经元“沉默”，但通道表达的启动需要数小时，而且不易逆转。我们正在开发一种快速和可逆的方法来沉默单个神经元的活动，这涉及到通道的表达，这些通道经过化学修饰使它们对光敏感。由于闪光可以快速而准确地应用，这种方法允许更大的时间和空间控制。我们的光激活通道由可光异构化的小分子偶氮苯(AZO)的衍生物和Shaker K+通道组成。AZO衍生物在一端具有半胱氨酸反应性马来酰亚胺(MAL)基团，允许连接到摇床中的特定半胱氨酸， 另一端是阻孔剂四乙基铝(TEA)。在其拉长的反式形式中，MAL-AZO-TEA分子可以到达孔洞并堵塞，但在360 nm的光照射下，AZO光异构化为弯曲的顺式，这太短了。420 nm光的照射加速了顺式到反式的反向转化，恢复了受阻状态。因此，不同波长的照明会延长或收缩茶叶组，从而阻塞和解堵通道。为了最大限度地发挥通道对神经活动的影响，我们将在Shaker通道中引入突变，消除失活，并将其电压依赖的激活转变为超极化电位，使通道在其未被阻断的状态下结构性地活跃。我们将首先在非洲爪哇卵母细胞中表达该通道，并鉴定其对光的敏感性。然后，我们将把编码该通道的基因引入培养的哺乳动物神经元，应用修改后的AZO，并使用光来超极化和沉默电活动。最后，光激活通道将在完整视网膜的神经节细胞中表达。适当的光照应该改变动作电位的激发，即使在缺乏功能性视杆和视锥的视网膜中也是如此。光激活通道提供了一种准确和可逆的方式来调节神经活动，并为影响神经系统的实验和治疗目的打开了一个新的光-生物电子接口。

The ability to artificially control neuronal activity is important both experimentally, for understanding neural circuits, and therapeutically, for compensating for damage or degeneration of neural structures. Commonly used electrical or chemical methods of neural control are invasive and can be spatially and temporally inaccurate. Targeted expression of genes encoding K+or C1- channel has been used to "silence" specific neurons, but initiation of channel expression takes hours and is not easily reversible. We are developing a rapid and reversible method for silencing the activity of individual neurons that involves expression of channels that are chemically modified to render them light-sensitive. Because light flashes can be applied rapidly and accurately, this approach allows greater temporal and spatial control . Our light-activated channels consist of a derivative of the small photoisomerizable molecule azobenzene (AZO), and a Shaker K+ channel. The AZO derivative has a cysteine-reactive maleimide (MAL) group on one end, allowing attachment to a specific cysteine in Shaker, and a pore-blocking tetraethylalnmonium (TEA) group on the other end. In its elongated trans form, the MAL-AZO-TEA molecule can reach the pore and block, but upon exposure to 360 nm light, the AZO photoisomerizes to its bent cis form, which is too short. Illumination with 420 nm light accelerates the reverse cis to trans conversion, restoring the blocked state. Hence illumination with different wavelengths extends or retracts the TEA group, blocking and unblocking the channel. To maximize the impact of the channel on neural activity, we will introduce mutations in the Shaker channel that eliminate inactivation and shift its voltage-dependent activation to hyperpolarized potentials, making the channel constitutively active in its unblocked state. We will first express the channel in Xenopus oocytes and characterize light-sensitivity. We will then introduce the gene encoding the channel into cultured mammalian neurons, apply the modified AZO, and use light to hyperpolarize and silence electrical activity. Finally, light-activated channels will be expressed in ganglion cells in intact retina. Appropriate illumination should alter action potential firing, even in retina that are lacking functional rods and cones. Light-activated channels provide an accurate and reversible way to regulate neural activity and open a new opto-bioelectronic interface for influencing the nervous system for experimental and therapeutic purposes.