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INTRACELLULAR LOOP 1
HPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEG
(amino acids 139 thru 194)
Among members of the ABC transporter family, there is some conservation in the length and amino acid sequence of the loops. The transmembrane helices, except in certain positions, have less sequence similarity among ABC transporters. Structural prediction algorithms were used to infer that intracellular loops 1 and 2 have two major alpha-helical segments. The locations of the alpha helices composing the loops are also predicted to be similar among the two. Many ion channels have been shown to use their intracellular loops for gating, so it wouldn't be surprising if it turns out CFTR uses its intracellular loops for gating as well. It has been speculated that the intracellular loops of CFTR may be involved in passing information along to R-domain and pore.
E193K is a CF mutation which decreases the rate of channel opening .
Membrane proteins often have charged amino acids in their loops near the surface of the membrane. They may be involved in attracting permeant ions to the pore, or they may be there simply to keep the transmembrane helix segment from pulling the loop into the membrane.
Mutation I148T 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, A1067T, G1244E, S1255P, and G1349D.
The mutations E193K (this loop), 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
When comparing CFTRs of different species, it appears the the intracellular loops are generally more highly conserved than the extracellular loops or transmembrane helices of CFTR.
Intracellular loops appear to be critical for proper CFTR protien processing.
RESEARCH RESULTS:
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. Science 9/7/01, Vol 293 pgs 1793-1800
Mutations causing Cystic Fibrosis as well as in vitro mutagenesis indicate cytosolic loop 3 has subtle effects on channel conductance and therefore may be close to the pore. It has been postulated that some of these intracellular loops may interact with the cytosolic NBD and R domains. There are 3 known mutations in this loop that cause CF.
No cytoplasmic loop mutations affects ion conduction greatly. But when glycosylation sequences were engineered into this loop, there was incorrect protein processing (as well as in ICL3 and ICL4).
In the cardiac isoform of CFTR, exon 5 is lacking. Exon 5 codes for 30 amino acids in intracellular loop 1. This splice varient, when expressed in epithelial cells never makes it to the cell membrane, yet CFTR channel activity can be detected in cardiac tissue. When measuring the conductance of this isoform in intracellular vesicles, it shows non-wild type properties, including more subconductance state behavior (6 and 3pS) along with a reduced open probability to the wild type conductance of 8 pS. It can be concluded that loss of these 30 amino acids in intracellular loop 1 results in both mis-targeting to cell membrane of epithelial cells as well as the function of the channel.
Deletion of exon 5 (residues 163-193) results in a common varient of CFTR found in mouse epithelial tissue, but causes disruption of human CFTR due to both processing and function.
Generally, mutations introduced into 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.