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INTRACELLULAR LOOP 4
QTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMR
(amino acids 1035 thru 1102)
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 ICL2 in CFTR (ICD2 in MsbA) consists mostly of alpha-helical residues (amino acids 193 thru 207 and 237 thru 252). The electron densities for amino acids 208 to 236 were found to be diffuse within the crystal structure. Note that there is no direct counterpart of ICL4 from CFTR to MsbA, as MsbA functions as a homodimer and CFTR as a single protein. However, it could be inferred that ICL4 in CFTR corresponds to ICL2 in MsbA, as it is widely believed that CFTR arose as a duplication of a protein similar to MsbA at some point in the past. Science 9/7/01, Vol 293 pgs 1793-1800
Mutations in this fourth intracellular loop have been found to cause changes in gating behavior, but not pore selectivity for anions. Therefore it has been suggested that ICL4 probably has a function similar to the NBDs. These same mutations in ICL4 seem to effect the processing of the CFTR protein and therefore result in a large decrease in channel density at the cell surface.
The A1067T mutation is normally processed in epithelial cells, and is able to mediate normal chloride transport, however it is associated with CF, specifically pancreatic insufficiency. Other mutant CFTRs like this include G178R, I148T, G1244E, S1255P, and G1349D.
Q1035 thru R1102, when comparing this stretch of amino acids among CFTRs of various divergent species, appears to be the most highly conserved subdomain of the entire CFTR sequence.
CF-causing mutations occur in ICL4, including H1085R and R1066C. Gating behavior, rather than chloride conductance rate appears to be most affected by mutations in this loop. There is also a decrease in the overall response of CFTR to ATP and pyrophosphate. This strongly suggests that the intracellular loops, including ICL4, participate in interdomain interactions within the cytoplasm. One theory is that they help link the NBD domains with the transmembrane domains during channel gating.
RECENT RESEARCH:
ICL4 appears to be a mutational hotspot with many missense mutations (at least 19 known) in CF. The sequence of this loop is highly conserved among members of the ABC transporter family.
Naturally occurring mutations in this loop have been shown to affect protein processing but not to the degree affected in the deltaF508 mutation. No change in the conductance (rate of ion travel) thru the channel has been found, however. Gating of the pore is definitely affected. It's been speculated that the 4th intracellular loop is involved in coupling the activity of the NBDs to the pore.
When glycosylation sequences were engineered into this loop to show it is located on the intracellular side of the cell membrane, there was incorrect protein processing (the same thing happened with ICL3 and ICL1).
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