The Connexin Connection

Biologists are vigorously and enthusiastically exploring the structure and function of channels known as connexins, which link cells to one another. Connexin research is "hot," says one scientist heavily involved in the field, as investigators seek to answer a host of questions ranging from what passes through these channels to when and why they appear and disappear. Understanding how these channels work may also lead to improved drugs for diseases linked with connexin mutations. "From my perspective, what has made connexins hot is their association with a number of human diseases. We now have good evidence that mistakes in connexin genes underlie these disorders," says Alan Shiels, assistant professor of ophthalmology and genetics at Washington University. Shiels is studying the link between connexin mutations and inherited cataracts. Daniel Goodenough, professor of cell biology at Harvard Medical School, whose recent research points to a possible link between connexin mutations and female infertility, 1 notes another reason for activity in this area: The size of the family of connexin genes has not yet been determined. There are at least 14 mammalian genes, says Michael V.L. Bennett, professor of neuroscience at Albert Einstein College of Medicine. Connexins are known by numbers; for instance, connexin 26. The number refers to the molecular weight of the protein: 26,000 Da. Half a dozen connexins combine to form a connexon, which extends from a cell. The two connexons "dock" in the intercellular space. Shiels likens it to the joining of Europe's channel tunnel, half of which was built from England, half from France: The arrangement looks like half a dozen tubes bundled together. The full channel is 12 protein subunits. "You end up having hundreds and hundreds of these channels packed into a patch called a plaque," says Mark Yeager, associate professor of cell biology at the Scripps Research Institute and director of research in the Division of Cardiovascular Disease at the Scripps Clinic. The complete length of a channel, including the distance through each cell membrane and the space between cells, is about 250 angstroms, notes Yeager. The connexins are about 70 angstroms in diameter. Top: Surface expression of a gap junction protein, connexin 43. Bottom: Same field viewed with phase contrast optics shows cells, their nuclei, and borders between cells where junctions are located. Research Questions "The channels are believed to transmit certain biochemicals. What we don't know about the channels is what they transport. What are the signals, the biochemicals? We know they are small molecules," says Shiels, whose lab has identified two mutations of two connexin genes linked to inherited cataracts. 2,3 Inherited cataracts are congenital, resulting in blindness. (These are distinct from age-related cataracts.) "The lens is the most rich source of gap junctions [another term for connexins] in the human body," he says. In cell culture, Shiels has found that the mutant proteins fail to make the connexins. He assumes that whatever would travel through the channels keeps the lens clear. But determining what molecules pass through is "a technically daunting task," write Thomas White, instructor in neurobiology, and David Paul, professor of neurobiology at Harvard Medical School. 4 Beyond discovering what molecules traverse the connexins is the way that traffic is regulated. "The Holy Grail of membrane channel biology is to understand how channels open and close. If you understand [that], then you have insight into regulation. If you understand regulation, you can say something about disease and even pharmacology and treatment of disease," says Yeager, who recently described the structure of a membrane channel using electron crystallography. 5 He hopes to understand how mutations in connexins form, thus determining the impact of mutant protein in the connexin. Referring to the connexin mutations involved in nonsyndromic deafness (deafness not associated with any other symptoms), he says, "There are at least a couple of dozen mutations identified that have been associated with hearing loss. Some of these clearly block the channel into what we think is a closed state," Yeager says. Understanding the mutations will allow researchers to learn what parts of the protein are important for keeping the connexin open and functioning. Goodenough is exploring the role of connexins in development and possibly their role in female infertility, the outcome of unexpected findings in studies of knockout mice. "The knockout of one of the connexins, 37, resulted in female sterility. This opened up a whole interesting bidirectional communication that goes on between the developing oocyte and the surrounding nurse cells, called granulosa cells," he says. Previous research had shown communication between the two, but the findings from his lab demonstrated how complicated it is. "There's clearly quite an interesting conversation that is keeping these two different cell types apprised of each other's developmental stages as follicular genesis goes forward. At a very critical moment without this connexin there the conversation is interrupted, and the follicle fails," he remarks. The granulosa cells, however, interpret the communication gap as the occurrence of ovulation and mistakenly develop into corpa lutea. As is the case with other connexins, the identity of the molecules passing between the cells is unknown. But he plans in vitro experiments to determine what they might be. Another puzzle confronting connexin researchers is that connexins are not necessarily permanent structures. "It's clear that the cell can call up different members of the [connexin] family at different times, depending on developmental cues. They can come on briefly and go away after ostensibly serving some short communication function which is no longer needed," he says. "The complexity is mind boggling," he comments. Connexins pose another "conundrum," notes Bennett, using as an example mutations in connexins 26 and 32. These are known to lead to nonsyndromic deafness and a neuropathy known as Charcot-Marie-Tooth disease, respectively. Yet, he notes, the liver and the pancreas express both connexins, with no apparent ill effects in those organs. It may be that if there is a mutation in one of the connexins, the other takes over. "That's what everybody calls redundancy. That's all very well, but there has to be selection pressure for redundancy, or you just lose the gene over time. And it isn't clear what the selection pressure is when, if you don't have connexin 32 in your liver, you have a serious neuropathy, not a liver problem," he observes. Researchers are also probing the way connexins allow molecules to pass through them. Yeager notes that connexins have similarities to the two main categories of ion channels: regulation by voltage and by ligand. In connexins, "changes in intracellular pH and calcium concentration, as well as phosphorylation of the protein, can alter the conducting state," he says. A third method involves hydrophobic molecules that can permeate the cell membrane. "These molecules can interact with the channel and switch it into a closed state," he says. He notes that one such molecule, oleamide, has been identified. 6 But it's not known how such molecules block communication. Implications for Drugs Understanding in detail the nature of these mechanisms offers three potential sites for drugs to deal with connexin-related disorders, Yeager notes. "The single most important health issue is getting a better understanding and control of what happens to these communication circuits during ischemia around heart attacks," says Goodenough. Tissue damage resulting from a heart attack can alter the connections, he explains. Dead cells are spread among living ones, disrupting and blocking action potentials, he says. Drugs to remedy that would be very helpful for people recovering from heart attacks. But this is a long way off, as is the possibility of developing drugs to treat connexin-related deafness and neuropathies, since little is know about the mechanism behind those disorders. Knowledge of the connexin-disease link is still in its early days. "All of this is still in a very basic level," Yeager cautions. And there's more to deal with than just the early stages of the research. Studying connexins demands groups of cells, says Goodenough. "One of the things that's unusual about this field is that the functions [of connexins] are collective functions. You need groups of cells, and even groups of cells in tissue culture aren't right, because in tissue culture they are not mimicking what they do in an organ. One needs to be working with the whole animal as much as possible. I think this makes it a particularly challenging field to try to get at function," says Goodenough. References 1. A.M. Simon et al., "Female infertility in mice lacking connexin37," Nature, 385:525-9, 1997. 2. D. Mackay et al., "Connexin 46 mutations in autosomal dominant congenital cataract," American Journal of Human Genetics, 64:1357-64, May 1999. 3. A. Shiels et al., "A missense mutation in the human connexin50 gene underlies autosomal dominant zonular pulverulent cataract, on chromosome 1q," American Journal of Human Genetics, 62, 526-32, 1998. 4. T. White and D. Paul, "Genetic diseases and gene knockouts reveal diverse connexin functions" Annual Review of Physiology, 61, 283-310, 1999. 5. V. Unger et al., "Three dimensional structure of a recombinant gap junction membrane protein," Science, 283: 1176-80, 1999. 6. D.L. Boger et al., "Chemical requirements for inhibition of gap junction communication by the biologically active lipid oleamide," Proceedings of the National Academy of Sciences, 95:4810-5, 1998.

The Connexin Connection

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Harvey Black

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February 2000

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