Light-activated ion channels for neural control

人工控制神经元活动的能力在实验上对于理解神经回路和在治疗上对于补偿神经结构的损伤或退化都是重要的。通常使用的电或化学神经控制方法是侵入性的，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-通道已被用于“沉默”特定的神经元，但通道表达的起始需要数小时并且不容易逆转。我们正在开发一种快速和可逆的方法，用于沉默单个神经元的活性，该方法涉及经过化学修饰使其对光敏感的通道的表达。由于闪光可以快速准确地应用，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小的光致异构分子偶氮苯（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Shaker中的特定半胱氨酸， 和在另一端上的封孔四乙基铝（TEA）基团。在其伸长的反式形式中，MAL-AZO-TEA分子可以到达孔并封闭，但是在暴露于360 nm光时，AZO光异构化为其弯曲的顺式形式，其太短。用420 nm光照射加速逆顺式到反式转换，恢复阻断状态。因此，具有不同波长的照明延伸或缩回TEA基团，从而阻断和开放通道。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性激活转变为超极化电位，使通道在未阻断状态下组成性激活。我们将首先在非洲爪蟾卵母细胞中表达该通道并表征其光敏感性。然后，我们将编码通道的基因导入培养的哺乳动物神经元，应用修饰的AZO，并使用光来增强和沉默电活动。最后，光激活通道将在完整视网膜的神经节细胞中表达。适当的照明应该改变动作电位放电，即使在视网膜缺乏功能杆和锥。光激活通道提供了一种精确和可逆的方式来调节神经活动，并打开了一个新的光生物电接口，用于影响神经系统的实验和治疗目的。

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.