SAP97 regulates Kir2.3 channels by multiple mechanisms.
We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was approximately 13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 COOH-terminal domain and altering channel conformation.
Vikstrom
Karen L
KL
Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York, USA.
Vaidyanathan
Ravi
R
Levinsohn
Susan
S
O'Connell
Ryan P
RP
Qian
Yueming
Y
Crye
Mark
M
Mills
Jeffrey H
JH
Anumonwo
Justus M B
JM
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D006168:Guinea Pigs; D006325:Heart Atria / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D012756:Sheep; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; C501119:Dlg1 protein, rat; D008565:Membrane Proteins; D024661:Potassium Channels, Inwardly Rectifying