Modulation of Ca2+ entry and plasma membrane potential by human TRPM4b.
TRPM4b is a Ca(2+)-activated, voltage-dependent monovalent cation channel that has been shown to act as a negative regulator of Ca(2+) entry and to be involved in the generation of oscillations of Ca(2+) influx in Jurkat T-lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca(2+)-dependent inward and outward currents in whole cell patch clamp recordings. HEK-293 cells stably overexpressing TRPM4b showed higher ionomycin-activated Ca(2+) influx than wild-type cells. In addition, analysis of the membrane potential using the potentiometric dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and by current clamp experiments in the perforated patch configuration revealed a faster initial depolarization after activation of Ca(2+) entry with ionomycin. Furthermore, TRPM4b expression facilitated repolarization and thereby enhanced sustained Ca(2+) influx. In conclusion, in cells with a small negative membrane potential, such as HEK-293 cells, TRPM4b acts as a positive regulator of Ca(2+) entry.
Fliegert
Ralf
R
Calcium Signalling Group, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Glassmeier
Günter
G
Schmid
Frederike
F
Cornils
Kerstin
K
Genisyuerek
Selda
S
Harneit
Angelika
A
Schwarz
Jürgen R
JR
Guse
Andreas H
AH
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D002522:Chlorocebus aethiops; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D015220:Calcium Channels; D007476:Ionophores; D011993:Recombinant Fusion Proteins; D050053:TRPM Cation Channels; C437888:TRPM4 protein, human; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D002118:Calcium