ICl,swell is the main anion current responsible for RVD. It is known that the protein ICln is one of the main constituent/activators of the channel complex. Moreover ICln interacts with different cytosolic proteins like actin and the 4.1 protein (4.1R), a multifunctional protein that stabilizes cell shape and membrane mechanical properties.

We have already shown that ICln interacts in vivo with two different 4.1R isoforms (4.1R⁸⁰ and 4.1R¹³⁵), changing the subcellular localizations of both 4.1R proteins.

In this work we tried to elucidate the role of ICln-4.1R interaction, by analyzing the effects on ICl,swell activation and cell morphology.

By the use of a protocol of Correlative Light-Scanning Electron Microscopy, we have confirmed that the over-expression of 4.1R¹³⁵ (but not 4.1R⁸⁰) actually results in a significant increase in cell area and in the number of filopodia, while the co-expression with ICln reverts this effect.

By the use of the patch-clamp technique, in whole-cell configuration, we have seen that the over-expression of the 4.1R⁸⁰ (but not 4.1R¹³⁵) isoform in HEK cells leads to a significant increase of the IC,lswell activation, in hypotonic conditions.

Therefore, our results show that the two 4.1R isoforms play different functions in the cell.

Finally, ICln seems to act as a negative regulator of 4.1R function, by delocalizing it and counteracting the 4.1R¹³⁵ effect on cell morphology.