Researchers Report First Clone of A Calcium Channel Subunit Gene From An Invertebrate

BUFFALO, N.Y. -- University at Buffalo pharmacologists have cloned the gene that codes for a calcium-channel subunit in fruit flies and have discovered that the alpha-1 subunit bears a remarkable resemblance to one found in the rat brain.

The findings, to be published in the February issue of Journal of Neuroscience, may have immediate implications for the development of species-specific pesticides and new cardiovascular treatments.

They also are important in increasing the understanding of the variety of roles calcium channels play in vertebrates.

Support for the research came from Rhone-Poulenc, the French chemical company, the National Institutes of Health and the New York State chapter of the American Heart Association.

"Calcium channels from insects have different pharmacological properties from those of vertebrates," said Linda M. Hall, Ph.D., professor of biochemical pharmacology at UB and principal investigator. "Researchers have postulated that they therefore would have a very different molecular structure. But our work shows that they follow the same general architectural plan."

Calcium channels and the subunit molecules of which they are made are of interest for various reasons, Hall explained. They are ubiquitous in cells that process information by sending electrical signals in species ranging from Paramecium to humans, and they play a key role in skeletal muscle and heart function and are targets for numerous cardiovascular drugs. In addition, neuronal calcium channels are potential targets for neuroprotective agents for use in treating stroke, ischemia, head injury and other brain trauma.

Hall explained that calcium channels all have the same general "job," which is to open up when stimulated to allow calcium into the cell.

"However, the details of when they open up, how fast they open and close, and which other molecules affect their opening and closing vary from one channel type to another," she said.

"Since different channel types are found in different types of cells, such as heart, muscle, or neuronal cells, it would be a distinct advantage to be able to specifically open or block one type oock one type of channel at a time."channel is composed of five subunits, each of which, in turn, is a single protein molecule.

The fact that the structural similarity of the alpha-1 subunit has been conserved across such different species is a signal to researchers that the subunit and the calcium channel of which it is part perform an important function.

According to Hall, the regions of the protein that are identical between rats, humans and fruit flies, or Drosophila melanogaster, identify for scientists the functionally most important parts of the molecule, which, if altered, would disrupt channel functions.

"These sites are most important for understanding how channels open and close in the brain, which is how the brain sends electrical signals -- part of our normal thought processes -- from one region to another," said Hall.

Companies are interested in developing products, such as drugs or pesticides, which are specific for certain channel subunits. By targeting individual subunits that are in flies, for example, and not in humans, a pesticide could be effective without having a toxicological impact on humans.

"We now know which regions of the calcium channel are the same and which are different between the human and the fly," Hall said. "For species-specific pesticides, you want to target regions that are different."

Now that this channel subunit has been cloned, the researchers can develop systems for their expression outside the insect in tissue culture cell lines or in frogs' eggs.

"By expressing the channels in cell lines where they are not ordinarily expressed, pesticide researchers like those at Rhone-Poulenc can set up rapid and simple assays for testing compounds for insecticidal action against these channels," she said.

The research also has implications for the development of more targeted cardiovascular drugs, since the alpha-1 subunit contains the binding sites for several of them.

"One can design more effective drugs if you can pinpoint the molecular site of each binding interaction," said Hall. "Since the Drosophila channel subunit has the same general structural features as mammalian channel subunits, but different drug sensitivities, we can make chimeric channels constructed by splicing together different segments of equivalent subunits from mammals and fruit flies. We then test these chimeras to determine if their properties resemble those of flies or mammals. By systematically switching different parts of the channel, we can pinpoint regions that confer differences, and learn exactly where binding sites for specific drugs are located."

Now that Hall and her colleagues know the structures of several subunits in terms of their amino acid sequences, as well as the sequence of the different genes that encode them, they have a critical piece of information about the structure of the calcium channel in which these subunits are found.

"A major question in the calcium-channel literature is, 'Why are there so many different kinds of calcium channels?'" explained Hall. "Do the different kinds have overlapping or different functions?"

Channel subunit diversity comes from several factors. Different genes can code for the same general type of subunit, producing major structural variations. The messages encoded by each of these genes may also be spliced, or edited, in different ways, so that they encode slightly different proteins, each of which may have a different function.

Hall explained that with the new information, they can use Drosophila as a model system, and use genetic mutations of the subunit to inactivate one type of subunit at a time.

"We are using genetics like precise surgical tools to remove or functionally inactivate one subunit type at a time and observe the behavioral and developmental consequences," she said.

The work involves analyzing at a molecular level the genes that code for specific parts or subunits of calcium channels. By identifying the gene that codes for this particular subunit, the researchers are closer to discovering the fine details of its function, and can test its potential utility as a target for commercial products.