Coupling MAL-AZO-QA to the outside of Shaker adds a new light-switchable gate to the channel without compromising its normal voltage-dependent gating mechanism. By combining synthetic chemistry and protein mutagenesis, we have re-engineered the Shaker channel, adding an artificial control mechanism that is orthogonal to the natural one provided by evolution. This strategy may be extended to ionotropic or metabotropic receptors by incorporating specific receptor agonists or antagonists. Two conditions must be met for light-switchable ligands to alter the function of channels or receptors: (i) a given target protein must have a binding site for the ligand component of the molecule, and (ii) that site must be a precise distance from the covalent attachment site on the protein. MAL-AZO-QA attaches to a specific cysteine, but it might be possible to use other attachment strategies, including site-specific recognition sequences26
In the absence of MAL-AZO-QA, overexpression of the mutant Shaker channel may alter the normal electrical activity of hippocampal neurons. One would expect channel overexpression to hyperpolarize the membrane potential and suppress action potential firing, especially given the mutations that shift activation and reduce inactivation. Overexpression of Shaker channels can, however, result in a compensatory change in the expression of genes encoding native ion channels, causing hyperexcitability of neurons27
. In addition, overexpressed Shaker channels could form heteromultimers with native channel subunits, but this should occur slowly, as the turnover rate of native K+
channel subunits probably takes days. None of these perturbations should interfere with the use of light in regulating the activity of the MAL-AZO-QA-modified channels and ultimately in regulating neuronal firing. The procedure used here could, however, be modified to limit undesired effects of channel overexpression. For example, MAL-AZO-QA could be introduced at the onset of channel expression, ensuring that the channels are continually blocked, so that compensation is less likely to occur. Mutations resulting in milder changes in Shaker channel gating might also allow light regulation of firing without perturbing basal activity.
Site-directed functionalization of Shaker channels with MAL-AZO-QA introduces a new and permanent photoswitched gate. The power of this technique lies in its spatial and temporal accuracy, its noninvasiveness and its reversibility. The spatial resolution of channel activation should be limited only by the optics of the experimental system, and the kinetics of the response is limited only by the intensity of the light source.
Because these channels can control the firing of action potentials, we propose the name SPARK (synthetic photoisomerizable azobenzene-regulated K+
) channels. Irradiation of SPARK channels may be used to control neuronal populations, individual neurons, or even parts of neurons, such as axonal or dendritic branches. SPARK channels have the potential to transform the experimental analysis of neuronal circuitry not only in vitro
but also in vivo
. Shaker-type K+
channels can be readily expressed at high density in mammalian neurons using standard gene transfection techniques including lipofection28
, viral infection29
and transgenic gene expression27
. Amphipathic molecules similar to MAL-AZO-QA, such as voltage-sensitive dyes30
, penetrate deeply into neural tissue and label neuronal membranes. These features may enable the widespread use of SPARK channels, even deep in neural tissue. In addition to their use experimentally, SPARK channels may provide an effective means for controlling the activity of specific neurons downstream from sites of neural damage or degeneration. A particularly intriguing possibility is to use these channels to restore light-regulated activity in healthy retinal neurons after degeneration of rods and cones, the native photoreceptors. Light-regulated molecular machines, such as the SPARK channel, may also have applications in fields other than neurobiology, such as nanotechnology, bioelectronics and material sciences.