Electrophysiological Studies of Cftr Function: Effects of Cftr Mutations and Interactions With Other Chloride Channels (Acta Biomedica Lovaniensia, 236)

The interaction between CFTR mutants and/or polymorphisms results in different CFTR channel functions; which could explain why apparently normal CFTR genes cause disease. Both TM 12 and the intra-cytoplasmic stretch connecting TM 12 and NBD2 probably contribute to form the CFTR pore but may not contribute to anion selectivity.

The majority of the phosphorylation sites identified so far occur within the R domain. Phosphorylation of PKA sites present in this domain is the initial event in channel activation and the degree of phosphorylation determines the affinity of both nucleotide binding domains for ATP. One major goal of current research is to understand how phosphorylation of multiple R domain serines by PKA effectively regulates CFTR channel function.

Results obtained in this study, using constructs mutated at amino acids located in exon 13, and deletion constructs, indicated that the R domain indeed plays a crucial role in regulating the CFTR channel. The regulation efficiency is, at least in part, related to electric charge. Replacing the negatively charged glutamic acids to positively charged lysine at positions 822 and 826 in the C-terminal part of R domain resulted in an almost complete loss of CFTR channel activity. On the other hand, when a positively charged histidine was replaced by a more neutral amino acid at position 620 in the N-terminus of the R domain, CFTR channel activity was increased when compared with wildtype CFTR.

Neither of these mutants affected open pore properties of the channel, since the single channel conductance and selectivity were similar for all mutants and the wildtype CFTR. Deletion of a small fragment in the C-terminus of the R domain (amino acids 780-830) resulted in a complete loss of CFTR channel activity, which could be restored by replacement of that fragment by the mini MDR1 linker domain, which has a different amino acid sequence but similar number of amino acids and phosphorylation sites. This result implies the importance of C-terminal part of the R domain in controlling phosphorylation and CFTR channel activity.

In the study of interaction between CaCC and CFTR, expression of WT CFTR in CPAE cells significantly reduces the amplitude of ICl,Ca. The inhibition of ICl,Ca induced by agonist activation is further increased when CFTR channels were stimulated before activation of CaCC. Therefore, modulation of CaCC by CFTR is both activity-independent and activity-dependent. It is likely that the CFTR-CaCC interaction occurs at the plasma-membrane since the expression of DF508 CFTR does not influence CaCC. For a further understanding of the underlying mechanism of this interaction, a similar experiment was undertaken on CFTR hybrid constructs. The results showed that removal of the C- terminal part of the R-domain abolishes CFTR interaction with CaCC. Replacing the C-terminal part of the R-domain with the MDR1 linker domain rescued CFTR activity, but not the activity-independent inhibition of CaCC. These results do suggest that the C-terminal part of the R domain was involved in this channel-channel interaction.