Assessing the contributions of H-bonding donors to permeation rates and selectivity in self assembling peptides that form chloride selective pores

ORGN 39

John M. Tomich, jtomich@ksu.edu, Shawnalea J. Frazier, sjf5555@ksu.edu, and Takeo Iwamoto, iwamoto@ksu.edu. Department of Biochemistry, Kansas State University, Manhattan, KS 66506
A series of single and double amino acid substitutions were made in NC-1130, a channel forming peptide derived from the transmembrane M2 segment of the alpha subunit of the human spinal cord glycine receptor (GlyR). This sequence self assembles to form homo oligomeric water-filled pores in phospholipid bilayers that are Cl- selective and gate in the msec time frame. NMR studies and computer modeling suggest an assembled pore structure contains rings of threonines at sequence positions 14 and 17 with the narrowest passage occurring at the latter position. Chloride ions are hydrogen bond acceptors; consequently it is hypothesized the hydroxyl function contributes strongly to ion throughput and/or ion selectivity. Residue replacements in the peptide involving the 14th and 17th positions were designed to correlate hydrogen-bonding strength with selectivity and permeation rates. The replacements include cysteine, serine, homoserine, and diaminopropionic acid. All sequences were synthesized, purified and characterized by mass spectrometry. Circular dichroism was used to analyze for structural alterations. Electrophysiological studies in both MDCK monolayers and bilayers were employed to determine permeation rates and anion selectivity, respectively. The hydrogen bonding strengths of the –SH, –OH, and –NH functionalities correlate directly with anion selectivity and inversely with transport rates for the Cl- anion. These results will help in optimizing these two counteracting channel properties. A design introducing multiple weak binding residues will be discussed.
 

Membrane Active, Synthetic Organic Compounds
1:30 PM-5:05 PM, Sunday, 28 August 2005 Washington DC Convention Center -- Ballroom C, Oral

Division of Organic Chemistry

The 230th ACS National Meeting, in Washington, DC, Aug 28-Sept 1, 2005