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Ion Channel Malfunctions in Form of Retinitis Pigmentosa


HHMI research may help explain what causes the death of rod photoreceptors in the eye.

HHMI researchers have identified a molecular malfunction that causes a form of retinitis pigmentosa (RP), an inherited disease that causes progressive loss of vision and ultimately blindness.

Mutations in any one of 19 genes can cause RP. Although the researchers studied a specific mutation that alters only one of the 19 genes, their studies may offer a broad explanation for how the rod photoreceptor cells in the eye slowly die, which can ultimately lead to blindness.

The more we know about the fundamental processes that underlie these diseases, the closer we are to being able to intervene in an intelligent way.

William N. Zagotta

The studies were reported in an article published in the April 11, 2002, issue of the journal Neuron by Howard Hughes Medical Institute investigator William N. Zagotta and lead author Matthew C. Trudeau, both of whom are at the University of Washington Medical School.

Trudeau and Zagotta focused on a form of RP caused by a defect in the gene that codes for CNGA1, a subunit of an ion channel protein that normally shuttles to the surface membrane of light-detecting rod cells in the eye. Under normal conditions, CNGA1 co-assembles with another subunit, CNGB1, to form a channel complex that is comprised of four subunits. After the channel is assembled, it migrates to the surface of the rod cell, where it helps to translate light signals into neural signals that are transmitted to the brain. This channel closes when a light signal is detected, and this helps trigger a nerve impulse.

The researchers expressed the mutant CNGA1 subunit (called CNGA1-RP) that is involved in RP in frog eggs to probe how it malfunctions. To their surprise, the experiments showed that when the CNGA1-RP protein was expressed in frog eggs, it combined with other CNGA1-RP proteins, and seemed to function normally. However when it combined with CNGB1, no functional channels were expressed at the surface of the membrane.

To get a closer look at what was happening with the subunit, they attached a yellow fluorescent protein to both normal CNGA1 and to CNGA1-RP. The fluorescent protein permitted the researchers to see that CNGA1-RP failed to reach the plasma membrane surface when combined with CNGB1, even though the subunit proteins seemed to be produced normally and combine into channels in the cells.

“Since we knew that the CNGA1-RP was truncated at one end (the C-terminal region of the protein), we then reasoned that this truncation somehow affected the interaction with the CNGB1 subunit and prevented normal transport,” said Zagotta. The scientists reasoned that this truncated region likely interacted with the other end of CNGB1 in a region of the protein called the N-terminus.

To pinpoint which regions of the two subunits were interacting abnormally, the scientists used a “bait” and “fish” technique to detect molecular interactions between the two protein subunits. Basically, they created bait by attaching certain pieces of the subunits to tiny beads, which they then used to fish out the interacting pieces of the subunit.

After numerous experiments, the researchers found that the truncation in CNGA1-RP left a small stretch of CNGB1 on the N-terminal end uncovered. The researchers hypothesize that this small region of CNGB1 apparently functions as a regulatory signal that tells the cell not to allow the channel to be transported to the membrane surface.

Trudeau and Zagotta proved that this regulatory signal was crucial for transport by producing CNGB1 without the key regulatory signal. Even when combined with CNGA1-RP, this altered channel migrated normally to the membrane surface.

“So these experiments told us that the purpose of the normal interaction between CNGA1 and CNGB1 was to hide this trafficking signal,” said Zagotta. “While this is an unusual interaction, other laboratories have found similar interactions between subunits in other channels.

“Typically, these kinds of signals consist of amino acid sequences in proteins that tell the cell to keep a protein in the endoplasmic reticulum, where it is produced. While these signals are more commonly found on proteins that are supposed to be retained, sometimes other proteins have co-opted the signal to increase the fidelity by which these proteins are produced,” said Zagotta.

Understanding how CNGA1-RP malfunctions might well offer a clue to how rod cells are killed by the genetic defect, said Zagotta. “There are many ways such a defect might cause the cells to die,” he said. “One likely mechanism is that the absence of these channels—since they close in response to light—might lead the rod cell to think it is seeing light constantly. This malfunction might trigger a pathological state in the cells when they think they’re being exposed to light constantly from day one.

“Another possible cause of rod cell death is that the buildup of these proteins in the cell basically gums up the endoplasmic reticulum, ultimately killing the cell.”

Although the new finding will not likely lead to treatments for RP, Zagotta said “it does illustrate the general principle that the more we know about the fundamental processes that underlie these diseases, the closer we are to being able to intervene in an intelligent way. And in this particular case, nobody would have dreamed that the underlying cause of this form of RP was such a strange interaction between the subunits of a channel.”