title: Functional differences between TRPC4 splice variants.
abstract: Functional characterizations of heterologously expressed TRPC4 have revealed diverse regulatory mechanisms and permeation properties. We aimed to clarify whether these differences result from different species and splice variants used for heterologous expression. Like the murine beta splice variant, rat and human TRPC4beta both formed receptor-regulated cation channels when expressed in HEK293 cells. In contrast, human TRPC4alpha was poorly activated by stimulation of an H(1) histamine receptor. This was not due to reduced expression or plasma membrane targeting, because fluorescent TRPC4alpha fusion proteins were correctly inserted in the plasma membrane. Furthermore, currents through both human TRPC4alpha and TRPC4beta had similar current-voltage relationships and single channel conductances. To analyze the assembly of transient receptor potential channel subunits in functional pore complexes in living cells, a fluorescence resonance energy transfer (FRET) approach was used. TRPC4alpha and TRPC4beta homomultimers exhibited robust FRET signals. Furthermore, coexpressed TRPC4alpha and TRPC4beta subunits formed heteromultimers exhibiting comparable FRET signals. To promote variable heteromultimer assemblies, TRPC4alpha/TRPC4beta were coexpressed at different molar ratios. TRPC4beta was inhibited in the presence of TRPC4alpha with a cooperativity higher than 2, indicating a dominant negative effect of TRPC4alpha subunits in heteromultimeric TRPC4 channel complexes. Finally, C-terminal truncation of human TRPC4alpha fully restored the channel activity. Thus, TRPC4beta subunits form a receptor-dependently regulated homomultimeric channel across various species, whereas TRPC4alpha contains a C-terminal autoinhibitory domain that may require additional regulatory mechanisms.
authors.affiliation: Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany. schae@zedat.fu-berlin.de
authors.forename: Michael
authors.initials: M
authors.lastname: Schaefer
authors.forename: Tim D
authors.initials: TD
authors.lastname: Plant
authors.forename: Nicole
authors.initials: N
authors.lastname: Stresow
authors.forename: Nadine
authors.initials: N
authors.lastname: Albrecht
authors.forename: Gunter
authors.initials: G
authors.lastname: Schultz
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D014644:Genetic Variation; D049452:Green Fluorescent Proteins; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
chemical_list: D015220:Calcium Channels; D007473:Ion Channels; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019284:Thapsigargin; D006632:Histamine; D002118:Calcium