Abstract 949: Sap97 Regulates Kir2.3 Channels By Multiple Mechanisms
The inwardly rectifying potassium channel, Kir2.3, is a prominent component of the atrial inward rectification mechanism and preferentially localizes to the intercalated disc. The scaffolding protein, synapse associated protein (SAP97), is also found concentrated at the intercalated disk and interacts with Kir channel proteins through the channel protein’s c-terminal (CT) residues (SAI). We tested the hypothesis that SAP97 modulates whole-cell Kir2.3 currents by altering one or more biophysical properties of the underlying channel, and that this modulation requires the CT SAI motif.
Methods: Kir2.3 (wtKir2.3 and Kir2.3ΔSAI) and SAP97 were expressed in HEK293 cells singly and in combination. A combination of techniques (cell surface labeling, whole cell and single channel recordings) was used to investigate SAP97 effects Kir2.3 currents.
Results: Co-expression of wtKir2.3/SAP97, but not Kir2.3ΔSAI/SAP97, caused a ~2 fold increase in current density. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with only ~3% of Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3 leading to prominent co-localization of Kir2.3 and SAP97 and a ~40% increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was ~13 pS, while co-expression of SAP97 led to a wide distribution of unitary events with three distinct peaks centered at 16 pS, 29 pS and 42 pS. These changes occurred without altering channel open probability or whole-cell current rectification properties. Summary and
Conclusions: Association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance and these effects require an intact Kir2.3 CT domain. However, changes in single channel conductance play the major role in determining SAP97 effect on whole-cell Kir2.3 currents in this model system.