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1 Biology and Biochemistry, University of Houston, Houston, Texas, United States
* To whom correspondence should be addressed. E-mail: sdryer{at}uh.edu.
We carried out a yeast two-hybrid screen to identify proteins that interact with large conductance (BKCa-type) Ca2+-activated K+ channels encoded by the Slo1 gene. Nephrin, an essential adhesion and scaffolding molecule expressed in podocytes, emerged in this screen. The Slo1-nephrin interaction was confirmed by co-immunoprecipitation from brain and kidney, and from HEK293T cells expressing both proteins, and by GST pull-down assays. We detected nephrin binding to the Slo1VEDEC splice variant, which is typically retained in intracellular stores, and to the
4 subunit. However, we did not detect significant binding of nephrin to the Slo1QEERL or Slo1EMVYR splice variants. Co-expression of nephrin with Slo1VEDEC increased expression of functional BKCa channels on the surface of HEK293T cells, but did not affect steady-state surface expression of the other C-terminal Slo1 variants. Nephrin did not affect the kinetics or voltage-dependence of channel activation in HEK293T cells expressing Slo1. Stimulation of Slo1VEDEC surface expression in HEK293T cells was also observed by co-expressing a small construct encoding only the distal C-terminal domains of nephrin that interact with Slo1. Reduction of endogenous nephrin expression by application of siRNA to differentiated cells of an immortalized podocyte cell line markedly reduced the steady-state surface expression of Slo1 as assessed by electrophysiology and cell-surface biotinylation assays. Nephrin therefore plays a role in organizing the surface expression of ion channel proteins in podocytes, and may play a role in outside-in signaling to allow podocytes to adapt to mechanical or neurohumoral stimuli originating in neighboring cells.
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