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Meeting Abstract
This project uses the consequences of nature’s mutations – the source of evolutionary change – as a way to understand ion channel structure and function. Inwardly rectifying potassium (Kir) channels are critically to the control of cellular resting membrane potential and excitability. In vertebrate species, all Kir channels are regulated by the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2). We used Kir channels cloned from the sponge, Amphimedon queenslandica, a valuable model organism, as a way to understand PIP2 regulation of Kir channels. Using patch clamp experiments, sponge Kir currents decreased over time following patch excision. In two electrode voltage clamp, co-expression and activation of a voltage-sensitive phosphatase led to rapid decreases in sponge Kir current and wortmannin pre-treatment to lower endogenous PIP2 levels significantly reduced sponge Kir current amplitudes. While these are characteristics of the PIP2-dependence of vertebrate Kir channels, direct application of the lipid to the cytosolic side of membrane patches could not reactivate the sponge Kir currents. However, mutagenic substitution of two residues in the sponge Kir channel restored high affinity PIP2 reactivation of Kir currents in excised patches. The functional impact of these experimental mutations which recapitulate nature’s changes in the channel’s structure can be explained using a homology model of the sponge Kir channel. This research helps resolve the protein/phospholipid interactions required for Kir channel activation and how a phospholipid binding pocket evolved specificity for PIP2, as observed in vertebrate Kir channels.