Novel Ion Channels Can Be Characterized by a Combination of Oocyte Expression and Patch Clamping

Cloning of human disease-causing genes and sequencing of the human genome have identified many genes encoding putative channel proteins, including 67 putative K+ channel proteins. One way of characterizing the function of these proteins is to transcribe a cloned cDNA in a cell-free system to produce the corresponding mRNA. Injection of this mRNA into frog oocytes and patch-clamp measurements on the newly synthesized channel protein can often reveal its function (Figure 7-19). This experimental approach is especially useful because frog oocytes normally do not express any channel proteins, so only the channel under study is

▲ EXPERIMENTAL FIGURE 7-19 Oocyte expression assay is useful in comparing the function of normal and mutant forms of a channel protein. A follicular frog oocyte is first treated with collagenase to remove the surrounding follicle cells, leaving a denuded oocyte, which is microinjected with mRNA encoding the channel protein under study. [Adapted from T P Smith, 1988, Trends Neurosci. 11:250.]

present in the membrane. In addition, because of the large size of frog oocytes, patch-clamping studies are technically easier to perform on them than on smaller cells.

This approach has provided insight into the underlying defect in polycystic kidney disease, the most common single-gene disorder leading to kidney failure. Mutations in either of two proteins, PKD1 or PKD2, produce the clinical symptoms of polycystic kidney disease in which fluid-filled cysts accumulate throughout the organ. The amino acid sequence of PDK2 is consistent with its being an ion-channel protein, and it contains a conserved P segment. When expressed in oocytes, PDK2 mediates transport of Na+, K+, and Ca2+ ions. In contrast, the sequence of PKD1 differs substantially from that of channel proteins, and it has a long extracellular domain that probably binds to a component of the extracellular matrix. Coexpression of PKD1 with PKD2 in frog oocyte eggs modifies the cation-transporting activity of PDK2. These findings provided the first, albeit partial, molecular understanding of cyst formation characteristic of polycystic kidney disease and also suggest that some channel proteins may be regulated in complex ways. Indeed, most Na+ and K+ channel proteins are associated with other transmembrane or cytosolic proteins that are thought to regulate their opening, closing, or ion conductivity. I

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