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To identify anion binding sites in the pore of the cystic fibrosis transmembrane conductance regulator (CFTR), we made use of the ability of thiocyanate (SCN-) binding to block chloride current through the channel. We compared the conductance properties of wild-type CFTR (wt-CFTR) channels to channels carrying mutations in transmembrane (TM) segment 6: T338A, S341A, and T339A. SCN- binding affinity proved to be sensitive to alanine substitutions at T338 and S341, consistent with the notion that TM6 contributes directly to the interior structure of the CFTR pore.
Cystic Fibrosis is an autosomal recessive lethal disorder caused by a mutation in the chloride-selective ion channel CFTR. It is still not fully known which portions of the CFTR protein contribute to the pore structure, or which amino acid residues are directly involved in ion binding within the pore. However, it is known that ion selectivity characteristics and entry into the channel are related to the energy required to dehydrate the anion. The CFTR channel is proposed to form an integral membrane protein including 12 transmembrane alpha-helices, each divided into 2 domains: TMs 1-6, and TMs 7-12. The unique properties of TM6 lead us to believe it is part of the architecture of the CFTR pore. Physiological changes in TM6 configuration, achieved by substitution mutations of certain amino acid residues, led to alterations of anion conductance through the pore and indicated that some portion(s) of TM6 play a role in anion binding. The goal of the present study is to confirm and further identify TM6 amino acids that line the pore and contribute to anion-selectivity. This was done by using the most reliable probe of CFTR: a permeant anion. Thiocyanate (SCN-) is one such anion that is highly permeable (more so than Cl-), yet binds extremely tightly inside the pore, slowing conduction rates. By investigating the interactions between SCN- and the inside of the pore, it will be possible to determine further structure and characteristics of the CFTR channel which may prove beneficial in the ultimate treatment of cystic fibrosis.
Oocyte Preparation: Oocytes were extracted from adult female Xenopus and incubated in L-15 media at 18¡C before injections. Wild-type CFTR cRNA, along with T338A, T339A, and S341A cRNA were injected into oocytes: 50 nL per ooctye. Each oocyte also received cRNA for the ?2-AR, which is activated by isoproterenol. Experiments were recorded 42-96 hours after injection, discarding each oocyte after use.
Electrophysiology Procedures: Data aquisition was done with the two-electrode voltage clamp (TEVC) technique with pCLAMP8.0 software controlling the membrane potential. Normal bathing solution included a standard Cl- solution: ND96 (96mM NaCl, 2mM KCl, 1mM MgCl2, 5mM HEPES, pH 7.5). Thiocyanate solutions varied in concentrations ranging from 100µM to 20mM, in each of which SCN- was added to the ND96 solution. Oocytes were placed in a running bath of ND96, impaled with two borosilicate glass electrodes, and exposed to anion solutions subsequently, each exposure being followed by a wash of ND96. Exposures to SCN- solutions were kept to ~1 minute, while exposure to ND96 was ~3 minutes during activation. Two exposures of each solution were applied to each oocyte: one during the background recording and one during activation. CFTR channel-activation was accomplished using isoproterenol (ISO) solution (1-5 uM ISO). Three separate protocols were applied for each solution change: Step30 (membrane potential is stepped for 75 ms to a range of potentials between -140mV and +80mV); Ramp-Up (membrane potential ramped from -80mV to +60mV over a period of 200 ms); and Ramp-Down (membrane potential ramped from +60mV to -80mV over a period of 200 ms). Only Ramp-Down data was used in analysis for consistency reasons (known anion current).
The results of SCN interaction within the CFTR channel show that the mutation T338A significantly affected anion flow through the pore. The binding characteristic of thiocyanate along the wall of the pore lends more detail to the overall physical structure of CFTR and its importance in regulating ion currents.
Supported by the Howard Hughes Science Initiatives at Emory University (no. 52003071) , the Graduate Division of Biological and Biomedical Sciences, and the NSF (MCB-0077575)
Rob made use of the ability of the thiocyanate (SCN-) to identify anion binding sites inside the CFTR pore. The CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel is, when mutated, the main cause of the genetic disease Cystic Fibrosis. SCN binds tightly inside the pore at certain regions, which in turn block Cl current through the channel. Rob compared the conductance properties of wild-type CFTR channels to channels carrying mutations in transmembrane segment 6 of the proposed structure of CFTR. By using two-electrode voltage clamp, Rob was able to examine the effects of certain alanine substitution mutations on SCN binding. As expected, Rob found that mutations on the interior wall of the pore reduced anion binding, and therefore increased conductance through the pore. Further study indicated that only a few amino acids are directly involved in anion binding affinity.
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