Molecular and Functional Characterization of Hv1 Proton Channel in Human Granulocytes

Autor: Mónika Baráth, Anikó Rajki, Anna Orient, Éva Rajnavölgyi, István Kovács, Bence Rethi, Miklós Geiszt, Gábor L. Petheő, Arpad Lanyi
Rok vydání: 2010
Předmět:
Phagocyte
Neutrophils
Cellular differentiation
Immunology/Innate Immunity
Gene Expression
Ion Channels
Jurkat Cells
chemistry.chemical_compound
Superoxides
NADPH oxidase complex
Phagosomes
Chlorocebus aethiops
Elméleti orvostudományok
Cells
Cultured
Phagosome
Membrane Glycoproteins
Microscopy
Confocal
Multidisciplinary
NADPH oxidase
Reverse Transcriptase Polymerase Chain Reaction
Superoxide
Cell Differentiation
Orvostudományok
Respiratory burst
medicine.anatomical_structure
Biophysics/Membrane Proteins and Energy Transduction
COS Cells
NADPH Oxidase 2
Medicine
RNA Interference
Physiology/Immune Response
Research Article
Science
Blotting
Western
Biology
Cell Line
Tumor
medicine
Animals
Humans
NADPH Oxidases
Intracellular Membranes
Eosinophil
Molecular biology
Eosinophils
chemistry
Immunology/Leukocyte Activation
biology.protein
Protein Multimerization
Granulocytes
Zdroj: PLoS ONE
PLoS ONE, Vol 5, Iss 11, p e14081 (2010)
ISSN: 1932-6203
Popis: Voltage-gated proton current (I(Hv)) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of I(Hv) in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently H(v)1, a voltage-gated proton channel, was cloned, and leukocytes from H(v)1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about H(v)1 in human granulocytes. Using qPCR and a self made antibody, we detected a significant amount of H(v)1 in human eosinophil and neutrophil granulocytes and in PLB-985 leukemia cells. Using different crosslinking agents and detergents in reducing and non-reducing PAGE, significant expression of H(v)1 homodimers, but not that of higher-order multimers, could be detected in granulocytes. Results of subcellular fractionation and confocal imaging indicate that H(v)1 is resident in both plasmalemmal and granular membrane compartments of resting neutrophils. Furthermore, it is also demonstrated that H(v)1 accumulates in phagosome wall during zymosan engulfment together with, but independently of Nox2. During granulocytic differentiation early and parallel upregulation of H(v)1 and Nox2 expression was observed in PLB-985 cells. The upregulation of H(v)1 or Nox2 expression did not require the normal expression of the other molecule. Using RNA interference, we obtained strong correlation between H(v)1 expression and I(Hv) density in PLB-985 cells. It is also demonstrated that a massive reduction in H(v)1 expression can limit the Nox2 mediated superoxide production of PLB-985 granulocytes. In summary, beside monomers native H(v)1 forms stable proton channel dimer in resting and activated human granulocytes. The expression pattern of H(v)1 in granulocytes is optimized to support intense NADPH oxidase activity.
Databáze: OpenAIRE
Externí odkaz: