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SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism
Jodene Eldstrom1, Woo Sung Choi, David F Steele
1Department of Physiology, University of British Columbia, 2146 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada.
FEBS Letters
|July 16, 2003
Summary
The PDZ protein SAP97 enhances cardiac potassium channel hKv1.5 activity via an indirect N-terminal interaction, not direct binding. This novel mechanism differs from other Kv1 interactions, highlighting unique regulatory pathways for hKv1.5 function.
Area of Science:
- Molecular biology
- Cardiovascular physiology
- Ion channel function
Background:
- The voltage-gated potassium channel hKv1.5 is crucial for cardiac electrical activity.
- PDZ domain-containing proteins, like SAP97, often regulate ion channel function.
- Previous studies indicated SAP97 down-regulates Kv1 currents through C-terminal interactions.
Purpose of the Study:
- To investigate the functional interaction between hKv1.5 and SAP97.
- To elucidate the specific domains and mechanisms involved in this interaction.
- To determine if SAP97 influences hKv1.5 channel activity.
Main Methods:
- Site-directed mutagenesis of hKv1.5 N- and C-termini.
- Electrophysiological recordings (e.g., two-electrode voltage clamp) in Xenopus oocytes.
- Co-immunoprecipitation assays in HEK cells and rat ventricular myocytes.
- Yeast two-hybrid assays.
- Confocal microscopy for co-localization studies.
Main Results:
- SAP97 significantly increased hKv1.5 currents, contrasting with previous findings for other Kv1 channels.
- This increase was dependent on the Kv1.5 N-terminus; deletion abolished the effect.
- Deletion of the C-terminal PDZ binding motif had no impact on the SAP97-mediated current increase.
- No robust physical interaction or co-localization between hKv1.5 and SAP97 was detected in vivo or in vitro, except for inefficient co-immunoprecipitation in one experimental setting.
Conclusions:
- SAP97 enhances hKv1.5 potassium currents through an indirect mechanism involving the channel's N-terminus.
- This interaction pathway is distinct from previously described SAP97-Kv1 interactions.
- The findings suggest a novel regulatory mechanism for hKv1.5 channel function by SAP97, independent of direct physical binding.