PI(4,5)P 2 -dependence of GABA A receptor channel function revealed by optogenetic manipulation of a binding site
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Ionotropic GABA A receptors (GABA A Rs) mediate fast inhibitory neurotransmission in mammalian brains. While recent structural studies have identified that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], a well-established regulator of numerous ion channels, binds to the α1 subunits of GABA A Rs, the functional relevance of this binding has remained elusive. Here, we combine electrophysiology, molecular dynamics simulations, and a recently developed caged lysine technology to define the role of PI(4,5)P 2 in GABA A Rs. We show that GABA A Rs are insensitive to acute PI(4,5)P 2 depletions by voltage-sensing phosphatase, but sensitivity is conferred by neutralizing the K311 binding site, indicating high-affinity binding. Caging of K311 by use of genetic code expansion recapitulated phenotypes of K311 mutant, conferring sensitivity to PI(4,5)P 2 depletion, whereas uncaging restored insensitivity. Furthermore, caging K311 revealed decelerated activation, which then can be accelerated by uncaging. Additionally, PI(4,5)P 2 -dependence extends to glycine receptors, suggesting PI(4,5)P 2 is an important endogenous phospholipid modulator of inhibitory receptor channels.
