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Research Article

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

¹University of Houston

Submitted 6 August 2009 ; revised 21 September 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 multi-protein complexes in excitable and non-excitable 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 co-immunoprecipitation of endogenous proteins from differentiated cells of a podocyte cell line, 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 GST pull-down assays and by co-immunoprecipitation. Neph1 is partially co-localized in intracellular compartments with endogenous Slo1 in podocytes and ciliary ganglion neurons. Co-expression 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, 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; traffick; glomerulus; parasympathetic