Binding of the protein ICln to α-integrin contributes to the activation of IClswell current

Abstract

icl swell is the chloride current induced by cell swelling, and plays a fundamental role in several biological processes, including the regulatory volume decrease (RVD). ICln is a highly conserved, ubiquitously expressed and multifunctional protein involved in the activation of icl swell. In platelets, ICln binds to the intracellular domain of the integrin αIIb chain, however, whether the ICln/integrin interaction plays a role in RVD is not known. Here we show that a direct molecular interaction between ICln and the integrin α-chain is not restricted to platelets and involves highly conserved amino acid motifs. integrin α recruits ICln to the plasma membrane, thereby facilitating the activation of ICl swell during hypotonicity. Perturbation of the ICln/integrin interaction prevents the transposition of ICln towards the cell surface and, in parallel, impedes the activation of ICl swell. We suggest that the ICln/integrin interaction interface may represent a new molecular target enabling specific ICl swell suppression in pathological conditions when this current is deregulated or plays a detrimental role. Cellular volume changes occur in a variety of physiological processes, including transepithelial transport, cell migration, proliferation, and death. As limiting excessive or prolonged volume changes is essential for normal cell function and survival, most cell types are able to counteract volume perturbations by initiating the homeostatic processes of regulatory volume increase and decrease (RVI and RVD, respectively) 1-4. Activation of a chloride conductance upon cell swelling (ICl swell) is a key step in RVD and occurs via volume regulated anion channels (VRACs) 5,6. The mechanisms of osmosensing are complex and far from being completely understood. Several lines of evidence implicate integrins as upstream sensors of cell volume perturbations in mammalian cells after cell swelling or shrinkage 1,7-9. Integrins are a highly conserved family of heterodimeric adhesion molecules that consist of an αand a β-subunit and connect the extracellular matrix to intracellular signalling proteins and the cytoskeleton, and play a critical role in many vital cellular functions, such as cell adhesion, migration, invasion, differentiation, proliferation, apoptosis, growth-factor signalling and response to hypotonicity 10-13. By linking the exterior and interior space of cells, integrins permit a bidirectional transmission (outside-in and inside-out signalling) of mechanical and biochemical stimuli across the plasma membrane 14,15. Specifically, activation of integrins by hypotonicity 16 seems to trigger Cl − and organic osmolyte fluxes during RVD, possibly by recruitment of a member of the SRC family of kinases 17. Integrins also establish an intricate network of molecular interactions via their intracellular domains. The β-subunits are major players in this context, and bind signalling hubs such as talin and kindlins 18-20. The intracellular network of the α-subunits was less extensively studied. Evidence was gathered that the ...

Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells

“…It's more likely that the number of VRAC channels doesn't really change, and the chloride flow through the channels is modulated in another way. Translocation of the chloride ion current inducer protein, ICln, from the cytosol into the cell membrane for 10-20 minutes after initial hypotonic shock has been shown for various cells [13,44].…”

“…Volume regulated anion channel (VRAC) is a ubiquitously expressed chloride channel that has attracted much attention since the molecular structure of VRAC has been identified [4][5][6]. A growing body of evidence indicate that VRAC and their obligatory subunit, LRRC8A have critical roles in many cell functions including cell motility, proliferation, apoptosis, drug and metabolite transport, angiogenesis, and spermatid development, as well as in cell pathophysiological cell functions such as cancer drug resistance, ischemic brain edema, and glaucoma [7][8][9][10][11][12][13][14][15][16][17]. While the molecular structure of VRAC is well documented [18], understanding how VRAC expression at the membrane is regulated has been poorly explored due to lack of appropriate methodology.…”

O-GlcNAcylation Suppresses the Ion Current IClswell by Preventing the Binding of the Protein ICln to α-Integrin

“…Single cells expressing the transfection marker EGFP, dsRed, or both could be identified by fluorescence microscopy using FITC (excitation: 480/30x, dicroic: 505 dclp, and emission: 535/40 m) and TRITC (excitation: 540/25x, dicroic: 565 dclp, and emission: 605/55 m) filter sets. Selected cells were voltage-clamped using the wholecell patch-clamp technique as previously described (Gandini et al, 2008;Dossena et al, 2011;Tamma et al, 2011;Morabito et al, 2017;Schedlbauer et al, 2019). The resistance of the glass pipettes was 3-8 M when filled with the pipette solution (CsCl 125 mM, MgCl 2 5 mM, EGTA 11 mM, ATP Mg 2+ salt 2 mM, HEPES 10 mM, pH 7.2 adjusted with CsOH, and 300 mOsm/kg H 2 O).…”

“…Overexpression of the heterologous protein ICln up-regulates IClswell in several cell types (Paulmichl et al, 1992;Hubert et al, 2000;Furst et al, 2002b;Dossena et al, 2011;Schedlbauer et al, 2019) (Supplementary Figure S6). In these cells, the apparent IClswell is the sum of the endogenous IClswell and the ICln-induced IClswell.…”

“…During a hypotonic stimulus, ICln is transposed toward the plasma membrane and facilitates the activation of IClswell (Ritter et al, 2003;Dossena et al, 2011). Binding of cytosolic ICln to the intracellular domain of α-integrin is an essential prerequisite for ICln association with the plasma membrane and IClswell activation and requires a direct molecular interaction between highly conserved amino acid motifs of both proteins (Schedlbauer et al, 2019). Specifically, the sequence (61)ISLHA(65) (single-letter amino acid code; numbers refer to the amino acid position within the human ICln protein sequence), located in the β4-β5 loop and β5 sheet of the pleckstrin homology-like N-terminal portion of ICln (Furst et al, 2005), appears to be essential for binding to α-integrin as well as for the ICln-induced activation of IClswell.…”