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This Article Full Text Full Text (PDF) All Versions of this Article: 297/6/C1379    most recent ajpcell.00354.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Kim, E. Y. Articles by Dryer, S. E. PubMed PubMed Citation Articles by Kim, E. Y. Articles by Dryer, S. E.

Membrane Transporters, Ion Channels, and Pumps

Neph1 regulates steady-state surface expression of Slo1 Ca²⁺-activated K⁺ channels: different effects in embryonic neurons and podocytes

Department of Biology and Biochemistry, University of Houston, Houston, Texas

Submitted 6 August 2009 ; accepted in final form 23 September 2009

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels encoded by the Slo1 gene are often components of large multiprotein complexes in excitable and nonexcitable cells. Here we show that Slo1 proteins interact with Neph1, a member of the immunoglobulin superfamily expressed in slit diaphragm domains of podocytes and in vertebrate and invertebrate nervous systems. This interaction was established by reciprocal coimmunoprecipitation of endogenous proteins from differentiated cells of a podocyte cell line, from parasympathetic neurons of the embryonic chick ciliary ganglion, and from HEK293T cells heterologously expressing both proteins. Neph1 can interact with all three extreme COOH-terminal variants of Slo1 (Slo1_VEDEC, Slo1_QEERL, and Slo1_EMVYR) as ascertained by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation. Neph1 is partially colocalized in intracellular compartments with endogenous Slo1 in podocytes and ciliary ganglion neurons. Coexpression in HEK293T cells of Neph1 with any of the Slo1 extreme COOH-terminal splice variants suppresses their steady-state expression on the cell surface, as assessed by cell surface biotinylation assays, confocal microscopy, and whole cell recordings. Consistent with this, small interfering RNA (siRNA) knockdown of endogenous Neph1 in embryonic day 10 ciliary ganglion neurons causes an increase in steady-state surface expression of Slo1 and an increase in whole cell Ca²⁺-dependent K⁺ current. Surprisingly, a comparable Neph1 knockdown in podocytes causes a decrease in surface expression of Slo1 and a decrease in whole cell BK_Ca currents. In podocytes, Neph1 siRNA also caused a decrease in nephrin, even though the Neph1 siRNA had no sequence homology with nephrin. However, we could not detect nephrin in ciliary ganglion neurons.

potassium channels; slit diaphragm; traffic; renal glomerulus; parasympathetic neurons