title: Discrete control of TRPV4 channel function in the distal nephron by protein kinases A and C.
abstract: We have recently documented that the Ca(2+)-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca(2+) responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca(2+)]i imaging with immunofluorescence microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of the PKC pathway with phorbol 12-myristate 13-acetate significantly increased [Ca(2+)]i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with bisindolylmaleimide I diminished cellular responses to elevated flow. In contrast, activation of the PKA pathway with forskolin did not affect TRPV4-mediated [Ca(2+)]i responses to flow but markedly shifted the subcellular distribution of the channel toward the apical membrane. These actions were blocked with the specific PKA inhibitor H-89. Concomitant activation of the PKA and PKC cascades additively enhanced the amplitude of flow-induced [Ca(2+)]i responses and greatly increased basal [Ca(2+)]i levels, indicating constitutive TRPV4 activation. This effect was precluded by the selective TRPV4 antagonist HC-067047. Therefore, the functional status of the TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. Although the PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, the PKC-dependent pathway increases the activity of the channel on the plasma membrane.
authors.affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
authors.forename: Mykola
authors.initials: M
authors.lastname: Mamenko
authors.forename: Oleg L
authors.initials: OL
authors.lastname: Zaika
authors.forename: Nabila
authors.initials: N
authors.lastname: Boukelmoune
authors.forename: Jonathan
authors.initials: J
authors.lastname: Berrout
authors.forename: Roger G
authors.initials: RG
authors.lastname: O'Neil
authors.forename: Oleh
authors.initials: O
authors.lastname: Pochynyuk
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D016257:Fura-2 / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007685:Kidney Tubules, Collecting / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009025:Morpholines / Q000494:pharmacology; D009399:Nephrons / Q000187:drug effects / Q000378:metabolism*; D010477:Perfusion / Q000379:methods; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011758:Pyrroles / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C573308:HC-067047; D007211:Indoles; D007546:Isoquinolines; D008301:Maleimides; D009025:Morpholines; D010703:Phorbol Esters; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D013449:Sulfonamides; D050916:TRPV Cation Channels; C486576:Trpv4 protein, mouse; D005576:Colforsin; C011986:phorbol 12-tiglate 13-decanoate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium; D016257:Fura-2
keywords: Calcium Intracellular Release; Collecting Duct; Connecting Tubule; Mechanosensitivity; Membrane Trafficking; Protein Kinase A (PKA); Protein Kinase C (PKC); TRP Channels; [Ca2+]i Imaging