EGF Stimulates ICl_swell by a Redistribution of Proteins Involved in Cell Volume Regulation

Abstract

Background: ICln is a multifunctional protein involved in the generation of chloride currents activated during regulatory volume decrease (RVD) after cell swelling (ICl_swell). Growth factor receptors play a key role in different cellular processes and epidermal growth factor (EGF) regulates swelling-activated chloride permeability. Aim: We set out to investigate if the EGF-induced upregulation of ICl_swell could be explained by a rearrangement of ICln subcellular distribution and interaction with its molecular partners. Methods: NIH-3T3 fibroblasts were serum-deprived for 24 hours and stimulated with EGF (40 ng/ml) for 30 minutes. ICl_swell activation, ICln distribution and interaction with its molecular partner HSPC038 were assessed by whole cell patch clamp and fluorescence resonance energy transfer (FRET). Results: EGF treatment significantly enhanced the direct molecular interaction between ICln and HSPC038 and also resulted in an increase of ICln and HSPC038 association with the plasma membrane. Importantly, these events are associated with a significant increase of ICl_swell. Conclusions: The present data indicate that EGF might exert its role in the modulation of volume-sensitive chloride currents in part through activation and translocation of ICln and HSPC038 to the plasma membrane.

Crude venom from nematocysts of Pelagia noctiluca (Cnidaria: Scyphozoa) elicits a sodium conductance in the plasma membrane of mammalian cells

“…Hypotonic shock induced the activation of a large chloride current with the biophysical fingerprints of IClswell (outward rectification, slow voltage- and time-dependent inactivation at potentials higher than +40 mV; Fig. 2a, middle panels), similar to previous observations323334. The reversal potential of these currents is very close to 0 mV, i.e.…”

Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death

“…Cells were voltage clamped with the whole-cell patch-clamp technique and initially kept in hypertonic solution (see Materials and methods). In this condition, no obvious currents were detected (Fig 6A), according to previous observations [20,22,25]. Then, IClswell was activated following a 28% reduction (100 mOsm) of the extracellular osmolarity via omission of mannitol from the bath solution.…”

“…Patch clamp experiments were performed 48 hours post-transfection. Single transfected cells were selected by fluorescence microscopy and voltage clamped using the whole-cell patch clamp technique as previously described [20,22–25]. Pipette (125 mM CsCl, 5 mM MgCl 2 , 11 mM EGTA, 2 mM ATP-Mg ++ , 10 mM HEPES, pH 7.2 adjusted with CsOH, osmotic pressure 300 mOsm/Kg H2O adjusted with raffinose), hypertonic (125 mM NaCl, 2.5 mM CaCl 2 , 2.5 mM MgCl 2 , 10 mM HEPES, pH 7.4 adjusted with NaOH, osmotic pressure 360 mOsm/Kg H2O adjusted with mannitol), isotonic (125 mM NaCl, 2.5 mM CaCl 2 , 2.5 mM MgCl 2 , 10 mM HEPES, pH 7.4 adjusted with NaOH, osmotic pressure 300 mOsm/Kg H2O adjusted with mannitol) and hypotonic (125 mM NaCl, 2.5 mM CaCl 2 , 2.5 mM MgCl 2 , 10 mM HEPES, pH 7.4 adjusted with NaOH, osmotic pressure 260 mOsm/Kg H2O ) bath solutions were designed to isolate chloride currents.…”

Expression, purification and molecular analysis of the human ZNF706 protein

“…These factors are among the principal methods of gene expression regulation on eukaryotic cells [2]. Moreover, the ZNF706 gene was associated with the Epidermal Growth Factor (EGF) to control the cell volume via the ICln gene [3]. ZNF proteins are now known to have additional activities such as the recognition of RNA and other proteins [4].…”