Inwardly rectifying potassium (Kir) channels are critically important for regulating resting membrane potential in excitable cells, a job underscored by the severe pathophysiology associated with channel dysfunction. While all Kir channels require the activating lipid PIP2, many of these channels have diverse modulatory factors that couple to PIP2-dependent gating. Channels in the Kir3 (GIRK) family, in particular, have several co-activating elements, including G-protein betagamma subunits, ethanol, and sodium. During stimulation of Gq-coupled receptors, downstream activation of Protein Kinase C can phosphorylate and inhibit Kir3 channels, yet the mechanism of inhibition and phosphorylation sites are incompletely understood. We took a combined experimental and computational approach using neuronal Kir3.2 to investigate how phosphorylation at a putative PKC site identified in Kir3.1/3.4 could lead to channel inhibition. Kir3.2 inhibition was found to depend on the phosphorylation state of Ser-196, although mutagenesis data suggest it functions as an allosteric regulator of PKC inhibition. MD simulations identified a molecular switch whereby phosphorylation of Ser-196 recruits a critical gating residue, Arg-201, away from the sodium coordination site Asp-228. Neutralization of Ser-196 or Arg-201 resulted in less active channels which exhibited increased sensitivity to PKC inhibition. Additionally the interplay of PIP2 and PKC inhibition was examined in depth using homomeric Kir3.2, revealing that increases in channel-PIP2 interactions limit sensitivity to PKC inhibition, whereas low levels of PIP2 increase PKC sensitivity. Neutralization of Ser-196 uncoupled PKC inhibition from this PIP2 dependence. These studies suggest a model whereby PKC inhibition can occur along PIP2-dependent and PIP2-independent pathways, depending on the phosphorylation state of Ser-196.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-4213 |
| Date | 26 September 2013 |
| Creators | Adney, Scott |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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