High yield of #recombinant protein expression in #293F cells via transient transfection with #Hype-5 reagent

Modulation of TMEM16A Channel Activity by the Von Willebrand Factor Type A (VWA) Domain of the Calcium-Activated Chloride Channel Regulator 1 (CLCA1)

Monica Sala-Rabanal et al. JBC 2017.

ABSTRACT

Calcium-activated chloride channels (CaCCs) are key players in transepithelial ion transport and fluid secretion, smooth muscle constriction, neuronal excitability, and cell proliferation. The CaCC regulator 1 (CLCA1) modulates the activity of the CaCC TMEM16A/Anoctamin 1 (ANO1) by directly engaging the channel at the cell surface, but the exact mechanism is unknown. Here, we demonstrate that the von Willebrand factor type A (VWA) domain within the cleaved CLCA1 Nterminal fragment is necessary and sufficient for this interaction. TMEM16A protein levels on the cell surface were increased in HEK293T cells transfected with CLCA1 constructs containing the VWA domain, and TMEM16A-like currents were activated. Similar currents were evoked in cells exposed to secreted VWA domain alone, and these currents were significantly knocked down by TMEM16A siRNA. VWA-dependent TMEM16A modulation was not modified by the S357N mutation, a VWA domain polymorphism associated with more severe meconium ileus in cystic fibrosis (CF) patients. VWA-activated currents were significantly reduced in the absence of extracellular Mg2+, and mutation of residues within the conserved metal ion-dependent adhesion site (MIDAS) motif impaired the ability of VWA to potentiate TMEM16A activity, suggesting that CLCA1-TMEM16A interactions are Mg2+- and MIDAS-dependent. Increase in TMEM16A activity occurred within minutes of exposure to CLCA1 or after a short treatment with nocodazole, consistent with the hypothesis that CLCA1 stabilizes TMEM16A at the cell surface by preventing its internalization. Our study hints at the therapeutic potential of the selective activation of TMEM16A by the CLCA1 VWA domain in loss-of-function chloride channelopathies, such as CF.

Recombinant Expression of CLCA1 VWA

The VWA domain of CLCA1 was expressed in 293F cells via transient transfection with Hype-5 (OZ Biosciences, San Diego, CA) at 1:1.5 ratio (μg DNA: μl Hype-5), using 1 μg of plasmid per 1 million cells. Media from supernatants were harvested after 72 h. Protein was purified from media supernatant using Ni-NTA superflow resin (Qiagen, Hilden, Germany) and eluted in 5 ml of buffer A containing 50 mM K2HPO4 (pH 8), 300mM NaCl and 250 mM imidazole. Purified CLCA1 VWA was dialyzed into buffer B containing 20 mM HEPES and 150 mM NaCl (pH7.4), and concentrated in a centrifuge concentrator to 1 mM, calculated from absorbance at 280 nm.