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.
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