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. 2009 May-Jun;3(3):161-3.
doi: 10.4161/chan.3.3.8853. Epub 2009 May 27.

UNC80 functions as a scaffold for Src kinases in NALCN channel function

Haikun Wang  1 , Dejian Ren
Affiliations

UNC80 functions as a scaffold for Src kinases in NALCN channel function

Haikun Wang et al. Channels (Austin). 2009 May-Jun.

Abstract

Ion channels can be regulated by a wide spectrum of neurotransmitters and hormones, largely through G-protein-coupled receptors (GPCRs). G-protein-independent activation of ion channel currents by GPCRs has also been recorded, although the molecular identity of the channels and the activation mechanisms remain largely unknown. UNC80 is a protein that is associated with the NALCN Na(+) leak cation channel, and is required for the activation of this channel by the neuropeptide substance P through GPCRs in a G-protein-independent fashion. Here, we show that UNC80 binds Src kinases and recruits Src into the channel complex. This finding is consistent with the known requirement for Src kinases in the activation of NALCN, and may lead to new insights into the molecular mechanisms underlying G-protein-independent activation of the channel.

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Figures

Figure 1
Figure 1. UNC80 interacts with Src
Upper left panel: Total lysates from HEK293T cells transfected as indicated were immunoprecipitated with anti-Src (monoclonal, # SC-8056 from Santa Cruz) and blotted with anti-UNC80 , showing that wild-type (SrcWT) and activated Src (Src529), but not dominant negative Src (Src296), coimmunoprecipitated with UNC80. Upper right panel: In the reverse experiment, anti-UNC80 precipitated SrcWT and Src529, but not Src296. Lower panel: inputs were blotted with anti-Src showing that the antibody recognized both the wild-type and the mutant Src proteins.
Figure 2
Figure 2. UNC80 recruits Src into the NALCN channel complex
Lysates from HEK293T cells co-transfected with Flag-NALCN and Src, with or without UNC80, were immunoprecipitated using anti-Src antibody and then detected with anti-Flag. Src529 and SrcWT, but not Src296, caused NALCN to be precipitated by anti-Src antibody (lanes 2, 6, and 4, respectively). When cells were cotransfected with UNC80, a much larger amount of NALCN coimmunoprecipitated with Src529 and SrcWT (lanes 1 and 5).
Figure 3
Figure 3. The interaction is independent of tyrosine phosphorylation
HEK293T cells overexpressing Flag-NALCN, UNC80, and Src proteins were either untreated or treated with the SFK inhibitor PP1 (10 μM) for 2 h prior to immunoprecipitation with anti-UNC80 or anti-Src. Precipitates were probed with the indicated antibodies. UNC80, NALCN, Src, and Ig bands are indicated with arrows.
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