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INTRACELLULAR LOOP 3
RGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLT
(amino acids 933 thru 990)
Mutations in this intracellular loop are known to have subtle effects on conductance and rectification and it is therefore speculated that ICL3 is be close to where the pore is located in CFTR. There seem to be a large number of natural CF-causing mutations in the intracellular loops (including ICL3), as well as in NBD1. Mutations in this loop and loop 4 reduce open channel lifetime. It's possible this loop may be responsible for stabilizing the CFTR pore in the open conformation. They may interact with the NBD domains by coupling their catalytic activity to the transmembrane helices. Most of the amino acids composing the intracellular loops are hydrophilic, however not all of the loops are of the same overall length.
The only high-resolution structure of an ABC superfamily member (of which CFTR is a member) is for MsbA. This bacterial lipid transporter was crystallized by Chang and Roth and resolved to 4.5 Å resolution. It clearly shows the intracellular loops as having distinct alpha-helical structure, and the loop between TM6 and the NBD domain functioning as a bridge, or conduit, between the NBD domains and the transmembrane domains. This implies the intracellular loops are helping to transmit energy from ATP hydrolysis at the NBD domains into a structural change at the pore. Named the "intracellular domains" by the authors, the intracellular loop of MsbA corresponding to ICL1 in CFTR (ICD1) exists as three alpha-helices connected by short loops to form a "U"-like structure. And the second alpha-helix of ICD1 is highly conserved and is up against the NBD domain. The residues in MsbA composing ICD1 are from amino acids 111 to 121. Note that ICL4 in CFTR has no direct counterpart in MsbA, as MsbA functions as a homodimer, while CFTR is a single protein, however it could be inferred that ICL4 in CFTR is the mirror-image of ICL1 in CFTR because it is believed that CFTR formed by the duplication of an ancesteral ABC protein (similar to MsbA?) which was fused in frame to form a single protein. Science 9/7/01, Vol 293 pgs 1793-1800
RESEARCH RESULTS:
Point mutations in this loop have helped uncover the role ICL3 plays in channel conduction as well as regulation. It also affects intracellular processing (folding and targeting to cell membrane). The mutations S945L and G970R affect rectification, with a change to a weak outward recitfication.
There are at least 3 known CF-causing mutations in this loop.
There is a domain within ICL3 which resembles known cyclic nucleotide binding sites (catabolite-gene activating protein has one also). Mutations in it impair the ability of cGMP to activate CFTR without altering cAMP activation. This may be how cGMP is known to stimulate CFTR activity in vivo.
No known cytoplasmic loop mutation affects ion conduction greatly, however when glycosylation sequences were engineered into this loop, there was incorrect protein processing (as well as in ICL1 and ICL4).
Generally, mutations in the intracellular loops 1 and 2 tend to affect the pore such that there was an increased closed time, while mutations in intracellular loops 3 and 4 seem to be involved in decreasing the time the channel is open. Perhaps 1 and 2 help open the pore and 3 and 4 help keep it open. It's speculated that these loops help couple the NBDs to channel gating.
The mutations E193K, D648V, H949Y, and R1070Q are all CF-causing mutations that are associated with pancreatic sufficiency. They have no effect on chloride transport but reduce bicarbonate transport by 50-60 %. Nature 3/1/01 pgs 94-96