Regulation of podocyte BK(Ca) channels by synaptopodin, Rho, and actin microfilaments.
Mechanosensitive large-conductance Ca(2+)-activated K(+) channels encoded by the Slo1 gene (BK(Ca) channels) are expressed in podocytes. Here we show that BK(Ca) channels reciprocally coimmunoprecipitate with synaptopodin (Synpo) in mouse glomeruli, in mouse podocytes, and in a heterologous expression system (HEK293T cells) in which these proteins are transiently expressed. Synpo and Slo1 colocalize along the surface of the glomerular basement membrane in mouse glomeruli. Synpo interacts with BK(Ca) channels at COOH-terminal domains that overlap with an actin-binding domain on the channel molecule that is necessary for trafficking of BK(Ca) channels to the cell surface. Moreover, addition of exogenous beta-actin to mouse podocyte lysates reduces BK(Ca)-Synpo interactions. Coexpression of Synpo increases steady-state surface expression of BK(Ca) channels in HEK293T cells. However, Synpo does not affect the stability of cell surface BK(Ca) channels, suggesting a primary effect on the rate of forward trafficking, and Synpo coexpression does not affect BK(Ca) gating. Conversely, stable knockdown of Synpo expression in mouse podocyte cell lines reduces steady-state surface expression of BK(Ca) channels but does not affect total expression of BK(Ca) channels or their gating. The effects of Synpo on surface expression of BK(Ca) are blocked by inhibition of Rho signaling in HEK293T cells and in podocytes. Functional cell surface BK(Ca) channels in podocytes are also reduced by sustained (2 h) but not acute (15 min) depolymerization of actin with cytochalasin D. Synpo may regulate BK(Ca) channels through its effects on actin dynamics and by modulating interactions between BK(Ca) channels and regulatory proteins of the podocyte slit diaphragm.
Kim
Eun Young
EY
Department of Biology and Biochemistry, University of Houston, Texas 77204-5001, USA.
Suh
Jae Mi
JM
Chiu
Yu-Hsin
YH
Dryer
Stuart E
SE
D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000502:physiology*; D051379:Mice; D008840:Microfilament Proteins / Q000502:physiology*; D023421:Models, Animal; D050199:Podocytes / Q000166:cytology / Q000502:physiology*; D020741:rho GTP-Binding Proteins / Q000502:physiology*
D000199:Actins; C494841:KCNMA1 protein, human; C465449:Kcnma1 protein, mouse; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D008840:Microfilament Proteins; C503967:SYNPO protein, human; C503968:Synpo protein, mouse; D020741:rho GTP-Binding Proteins