C Terminus of Nucleotide Binding Domain 1 Contains Critical Features for Cystic Fibrosis Transmembrane Conductance Regulator Trafficking and Activation

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl؊ channel physiologically important in fluidtransporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest ␤ strands (␤ c 5 and ␤ c 6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the ␤ c 5-␤ c 6 hairpin, within the ␤ c 5 strand (H620Q), within the ␤-turn linking the two ␤ strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the ␤ c 6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.Mapping ligand binding sites within ionic channel structures is important to understand the mechanism of action of channel modulators (i.e. activators and inhibitors), to identify regions required for selective interaction with modulators, and to design more selective and more potent agents. Such mapping, called pharmaco-topology, has been performed for the sulfonylurea receptor (SUR) regulatory subunits of K ATP channels (reviewed in Ref.

Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

“…Significantly, S10 contains three unique hydrophobic aromatics in CFTR, but in all other ABC C family members, S10 has instead at least one strongly polar residue, primarily Glu, Arg, Lys, and Gln. This region of high conservation, namely S9 and S10, coincides with the hydrophobic region we have described here as the site of CFFT-001 binding immediately below helices H8 and H9 and is also the location of residues shown to affect channel activity, including His-620 (56). This high degree of CFTR-specific conservation supports the observation that this region plays a role in conformational changes related to CFTR function and/or processing.…”

“…This should promote NBD dimerization and lead to an enhanced open probability, increased channel activity for H620Q mutant channels and the potentiating effect of CFFT-001. It is possible that H620Q and CFFT-001 act to force an "unnatural" gating; however, the likelihood of this is low, considering that the portion of NBD1 affected appears to be a regulatory hot spot based on our sequence analysis and the observed effects of mutations here (56). Note that although phosphorylation is expected to have an overlapping effect to compound binding in similarly disrupting the R region:NBD1 interface, R region phosphorylation has numerous other effects including enhancement of R region interactions with other parts of CFTR (9) and modulations of R region binding with other binding partners.…”

“…Because the H620Q variant displays additional peak shifts relative to those observed for compound binding, there are certainly other consequences of the mutation. The Becq group (56) has demonstrated that the region surrounding H620 is critical for trafficking and gating. Although H620Q does not affect maturation/processing, an H620P mutation does, highlighting the importance of this position and the nature of the residue in it.…”

“…H620Q Variant Reduces Helicity of H8 and H9 at the C Terminus of F508del NBD1 ⌬RI⌬RE-To address possible conformational changes within NBD1 that may be relevant to channel activity and/or misfolding, we focused on the C-terminal ␤-strands (S9 and S10) which have been shown to affect processing, activity and pharmacology of CFTR (56). In particular, an H620Q variant (in S9) originally identified in CF patients has an increased P o in single channels (56 -58).…”

Potential sites of CFTR activation by tyrosine kinases

“…17,18 Further studies of tyrosine kinase regulation may provide a better understanding of R domain structure and the conformational changes associated with CFTR channel regulation.…”