Light - induced depolarization of neurons using a modified Shaker K + channel and a molecular

To trigger action potentials in neurons, most investigators use electrical or chemical stimulation. Here we describe an optical stimulation method based on semi-synthetic light-activated ion channels. These SPARK (Synthetic Photoisomerizable Azobenzene-Regulated K +) channels consist of a synthetic azobenzene-containing photoswitch and a genetically modified Shaker K + channel protein. SPARK channels with a wild-type selectivity filter elicit hyperpolarization and suppress action potential firing when activated by 390 nm light. A mutation in the pore converts the K +-selective Shaker channel into a non-selective cation channel. Activation of this modified channel with the same wavelength of light elicits depolarization of the membrane potential. Expression of these depolarizing SPARK channels in neurons allows light to rapidly and reversibly trigger action potential firing. Hence, hyperpolarizing and depolarizing SPARK channels provide a means for eliciting opposite effects on neurons in response to the same light stimulus. INTRODUCTION Neurons have ion channels that are directly activated by voltage, ligands, temperature, and mechanical forces, but none are known to be directly sensitive to light. Consequently, when experimenters want to elicit neuronal responses, they usually apply electrical stimuli, which entails the placement of electrodes into or onto neural tissue, or chemical stimuli, which involves perfusion devices that have limited spatial and temporal precision. The idea of using light to dissect neuronal circuitry through remote stimulation has been around for decades (Fork 1971), however only recently have several strategies been devised that allow light to control ion channels and therefore neuronal function (Kramer et al. 2005). Light triggers for ion channel control include natural photoreceptive proteins (Boyden et al. 2005; Melyan et al. 2005; Zemelman et al. 2002), and caged compounds, which release active neurotransmitters when photolyzed with light We recently introduced a third type of light trigger, a photoisomerizable tethered ligand (or " photoswitch "), which directly controls ion channel activity in a light-dependent manner (Banghart et al. 2004). This channel, termed SPARK (Synthetic Photoisomerizable Azobenzene Regulated K + channel) opens with short wavelength light (e.g. 380-390 nm), triggering a K +-selective current that hyperpolarizes the membrane potential. Long wavelength light (e.g. 500-505 nm) accelerates the closure of the channel and turns off the current, restoring the original membrane potential. Expression of this SPARK channel in neurons allows light to reversibly hyperpolarize, and therefore silence, action potential firing. Here we have modified the K + channel protein to make the pore non-selective in its permeability to cations, …