Mechanism of Action of the Photoswitch Molecule QAQ

Abstract

Controlling neuronal excitability with light has emerged as a powerful and widely used approach in Neuroscience. Optogenetic tools function by enabling light to activate exogenously expressed ion channels that over-power endogenous electrophysiogical processes.In contrast, we have introduced a non-genetic approach to conferring light sensitivity on neurons that involves photochemical tools that regulate endogenous ion channels. Specifically, the design of the molecule QAQ is built around an azobenzene photoswitch group flanked by two symmetric quaternary ammonium groups which confer the ability to intracellularly block voltage-gated sodium, potassium and calcium channels. Blocking can be regulated by rapid and reversible photoisomerization of the central azobenzene group from trans to cis configuration upon 380 or 500nm light illumination, respectively. QAQ in its trans configuration blocks ion conduction through open channels enabling light-sensitive silencing of neurons while conversion to the cis configuration relieves the block.Given its double-charged character, QAQ is membrane-impermeable but it can enter cells through TRPV1 or P2X7 receptors if they are active. Since TRPV1 is mainly expressed in dorsal root ganglia neurons and participates in the perception of various external stimuli and pain, QAQ functions as a local anesthetic inhibiting neuronal excitibitly after cell entry.In that context, we have demonstrated that QAQ can modify nociception after selective loading of active pain neurons. However, the precise biophysical mechanism of action of QAQ on voltage-gated channels remains unclear. Therefore, we conducted an extended biophysical characterization using excised inside-out patches to elucidate binding characteristics of both QAQ configurations on a reporter channel, the voltage-gated potassium channel Shaker. Experiments involving mutations of Shaker and monitoring of QAQ trapping upon gate closure revealed interactions of QAQ with the channel. In summary, we examined the state-dependent persistence of blocking and our findings lead to multi-state binding model.