Primary Structure of Ion Channels

What do ion channels look like? Although ion channel currents have been measured for many decades, only recently with advancements in molecular biology have their structures been determined in any detail. The goal of understanding ion channel structure is a biochemical one. Usually, the initial step is to determine the primary structure of the ion channel protein. Currently, this is achieved by first cloning the ion channel gene and then reading the protein sequence from its cDNA. The nicotinic acetylcholine receptor was the first ion channel to have its sequence identified through the work of Shosaku Numa and colleagues in 1982. They first purified the ion channel protein from the Torpedo electric ray and obtained small bits of protein sequence. Then they designed short oligonucleotides that corresponded to their protein sequence and hybridized them to a cDNA library that contained genes from Torpedo. In this way, they obtained the entire cDNA sequence and read the entire amino acid sequence of nAChR.

Ion channels have also been identified using genetic methods. Shaker, a voltage-gated potassium channel from Drosophila melanogaster, was cloned by Lily and Yuh Nung Jan and colleagues in 1987. Shaker derives its name from the behavior of mutant flies when exposed to ether and was long suspected to be an ion channel based on biophysical studies. Shaker had already been localized to a region of the X chromosome. To identify part of the gene sequence, the Jan group performed Southern blots on a series of Shaker mutant flies with chromosomal rearrangements. Then, they were able to obtain the entire Shaker sequence by probing a Drosophila cDNA library with their partial DNA sequence.

Once a member of an ion channel family has been identified, other members of the same family can be identified by sorting through DNA libraries for genes that have related sequences. What was in the past done with petri dishes and nitrocellulose membranes is now being done using computers. With the growing information collected from genome sequencing projects, it is increasingly common for new ion channel genes to be identified through the Internet. Once a putative ion channel sequence has been found, a relatively straightforward sequence of steps can be used to clone the gene by amplifying from a sample of genomic DNA.

A great deal of information about what the ion channel looks like can be obtained from the primary structure. For example, it is known that membrane proteins contain discrete stretches of hydrophobic amino acids that span the lipid bilayer. These transmembrane segments can be identified with computer programs that search the protein for long stretches of hydrophobic residues. Combined with other information, one can derive a model of the membrane topology of the ion channel. The models that are generated, however, can be imperfect. There are several experimental methods by which scientists can test whether a region of the protein lies on the extracellular or intracellular face of the membrane. One is to identify sites that are glycosylated since they are known to be present only on the extracellular parts of a protein. Another is to raise antibodies against specific stretches of the protein and determine whether the antibodies bind from the inside or the outside surface of the cell.

The membrane topography is a useful scheme for classifying ion channels (Fig. 7). For example, there are three classes of potassium channels. The gene for voltage-gated potassium channels has six transmembrane segments, numbered S1-S6, and another hydro-phobic region between S5 and S6 called the P region, which does not cross the plasma membrane. The P region contributes to the pore of the ion channel. The inward rectifier potassium channels have two transmembrane segments with a P region between M1 and M2. A third class of potassium channels is called two-pore channels because each gene contains two P regions. Two-pore channels have four transmembrane

Two Pore Channel

Figure 7 The membrane topology of ion channels. Voltage-gated sodium and calcium channels resemble four voltage-gated potassium channel subunits linked together. The nicotinic acetylcholine receptor and the glutamate receptor are both ligand-gated ion channels but have different membrane topologies. Two-pore channels look like two inward rectifiers fused together. Among ion channels, CFTR belongs to a unique class of membrane proteins.

Figure 7 The membrane topology of ion channels. Voltage-gated sodium and calcium channels resemble four voltage-gated potassium channel subunits linked together. The nicotinic acetylcholine receptor and the glutamate receptor are both ligand-gated ion channels but have different membrane topologies. Two-pore channels look like two inward rectifiers fused together. Among ion channels, CFTR belongs to a unique class of membrane proteins.

segments, M1-M4. The membrane topology also gives clues about the function of different parts of the ion channel. For example, the binding site for acetylcholine in nAChR would be limited to those regions that faced the extracellular side.

Sequence analysis of different ion channel families suggests that ion channels are evolutionarily related. Two-pore channel genes resemble two inward rectifier potassium channel genes linked together, and voltage-gated sodium channels and voltage-gated calcium channels resemble four voltage-gated potassium channel genes linked in tandem.

The cloning of ion channels has revolutionized the study of ion channels by allowing scientists to make changes in the amino acid sequence and then test them. One particular strategy of making mutations—making chimeric ion channels—has been especially powerful in determining ion channel structure and function. In one well-known example, Numa, Sakmann, and colleagues used this method to conclude that M2 in the nicotinic acetylcholine receptor is important for determining the conductance of nAChR. In this strategy, one starts with two related ion channels that have different properties. In this case, it was known that nAChR expressed with the d subunit from calf has a conductance of 65 pS, whereas the d subunit from Torpedo has a conductance of 87 pS. In order to identify the residues that were responsible, Numa and colleagues made hybrid genes by splicing parts of the gene for the calf d subunit to the Torpedo d subunit and measured their conductance. With successive chimeras that contained increasingly smaller parts of the Torpedo gene, they were able to determine that the M2 segment is the region that determines the difference in conductance between the calf and Torpedo d subunits.

This approach has been instrumental in assigning functional roles to parts of ion channels. Using this approach, it has been revealed that ion channels are modular in design (Fig. 8). One caveat, however, is that one cannot exclude the involvement of other regions of the gene since replacement of those regions would not be detected if they shared a similar sequence or function.

Our understanding of ion channels has facilitated efforts to find more ion channel genes in computer

Figure 8 The modular design of voltage-gated potassium channels. Regions of the gene perform separate functions. Some of these functions can be transferred to other ion channels by transplanting that region alone.

databases. The parts of the ion channel that are functionally important are more likely to be conserved through evolution; therefore, database searches that are weighted to these residues are more likely to find homologs that may otherwise have little sequence conservation. The completion of the genomes of organisms with nervous systems, such as the fruit fly, D. melanogaster, or the worm, Caenorhabditis elegans, has ushered in a new set of more complex questions to be addressed. What are all the ion channels in a nervous system or in an animal? Given the redundancy of ion channel genes, what is the minimum set of ion channels needed for a functional nervous system?

Was this article helpful?

0 0
All About Alzheimers

All About Alzheimers

The comprehensive new ebook All About Alzheimers puts everything into perspective. Youll gain insight and awareness into the disease. Learn how to maintain the patients emotional health. Discover tactics you can use to deal with constant life changes. Find out how counselors can help, and when they should intervene. Learn safety precautions that can protect you, your family and your loved one. All About Alzheimers will truly empower you.

Get My Free Ebook


Post a comment