By David N. Leff

A clogged drain in the plumbing can be a major household nuisance.

One treatment is the so-called plumber's helper, a rubber plunger that sucks out the obstruction. Another is caustic lye, an aggresive chemical that dissolves the blockage.

For the mucus-clogged, breath-impeding airways of cystic fibrosis sufferers, there are no such ready-made remedies. They must undergo frequent pounding and pummeling to loosen and expel the thick, viscous mucus that not only hinders breathing but serves as an eventually lethal culture medium for infectious pathogens.

The root cause of this trachea-and-lung-obstructing hallmark of cystic fibrosis (CF) is a subtler blockage -- the absence or malfunction of chloride ion channels in the membranes of epithelial cells. The guilty gene is CFTR -- the cystic fibrosis transmembrane conductance regulator.

Besides preventing fluid transport across the membranes of airway cells, mutation of that gene, which encodes those channel-forming proteins, also affects other vital organs, notably pancreas, intestine and sweat glands. Their cells are deprived of the passageways that allow the free two-way flow of essential electrically charged atoms.

Chloride secretion drives fluid secretion, which normally maintains airway mucus in a less viscous, more expellable state.

Restoring or replacing flawed CFTR function has been the object of several gene therapy trials, so far inconclusive.

Into this breach steps (cautiously) a team of Kansas biochemists and physiologists with a synthetic peptide that actually inserts a functioning chloride channel into epithelial cell membranes.

The report of their highly preliminary in vitro experiment appears in the May issue of the American Journal of Physiology, published May 13, 1997. Its title: "A synthetic peptide derived from glycine-gated Cl1-- channel induces transepithelial Cl1-- and fluid secretion."

Two rival institutions of higher learning, the University of Kansas Medical Center, in Kansas City, (KU) and Kansas State University (KS), in Manhattan, Kan., joined forces to create and test this putative corrective for the messed-up CFTR gene function.

The paper's first author, physiology graduate student Darren Wallace, told how the two teams went about creating their artificial chloride channel: "John Tomich, a biochemist at KS, took the sequence for the transmembrane region of this protein that goes through the lipid membrane of the cell, and synthesized it chemically.

"Then," Wallace continued, "we at KU took that synthetic peptide and demonstrated that it's capable of self-inserting into a monolayer of epithelial canine kidney cell membranes and forming channels that conduct chloride."

Brain Neurons Point Way To Artificial Channels

Tomich, as his point of departure, sequenced a stripped-down version of a chloride-channel-forming glycine receptor in brain neurons, and fitted the resulting 23-amino-acid peptide with four lysine molecules.

Adding these amino acids to the ends of the molecule, Wallace explained, "helped make it more water soluble. The peptide was so hydrophobic that we were unable to get it into aqueous solution, so we were having difficulty delivering it to the cells."

In solving this problem, they took inspiration from a component of bee venom, which also forms channels. "This compound, called nelittin," Wallace noted, "also has four positive charges on the carboxy terminus of its peptide."

Initially, the KU group tested the synthetic pore peptide on a monolayer of canine kidney epithelia, as reported in its journal paper.

"The peptide," Wallace recounted, "increased short-circuit current, which is a measure of ion transport, and it appeared to be chloride-selective. We demonstrated that it caused fluid transport across the epithelium, indicating that chloride secretion was occurring.

"Now," he said, "we're beginning to use primary cultures of epithelial cells obtained from mouse trachea. The airway cells -- trachea, bronchii, lungs -- are where CF patients have the most difficulty."

The team plans next to demonstrate an effect in an organized epithelium, such as intestine, from a knockout mouse model of CF, which lacks the CFTR gene.

"Thus far," Tomich said, "we are able to create a water-lined pore in the canine kidney cells. Measuring the chloride channel-forming and fluid secretion activity of this new molecule, we're seeing a lot of atoms -- 100 million atoms a second -- move through the membrane.

"We are not working with CF patients." he added. "It will be several years before a drug therapy based on the synthetic molecule can be developed and tested clinically."

Cystic fibrosis and polycystic kidney disease are the commonest lethal inherited afflictions in the Western world. CF strikes one in every 2,000 babies born to Caucasian parents. In the U.S. and Canada, estimates of CF cases are in the range of 30,000 to 35,000. Even with improved clinical care, most die by their early 30s, with mortality of 1,500 a year. Death usually results from infection by the bacteria entrapped in the thick, sluggish mucus of the airways.

Besides CF, the Kansans' other focus of research is autosomal dominant polycystic kidney disease, where, Wallace pointed out, "there's an increase in electrolyte fluid transport across the renal epithelial cells -- in effect, the opposite of CF."

Patent Pending, Kansans Seek Commercial Partner

KU physiologist Lawrence Sullivan, the paper's senior author, told BioWorld Today: "We have a use patent application in, protecting our method of using the brain protein to form chloride channels." Their filing is headed, "A synthetic macromolecular channel assembly for transport of chloride ions through epithelium useful in treating cystic fibrosis."

Sullivan added: "The other thing is that we are interested in finding a pharmaceutical or biotechnology company that might be willing to support additional research on the peptide, with a view toward eventually commercializing it."

He concluded: "I realize, of course, that's far into the future yet." *