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Unformatted text preview: ferent combinations of exons, then different mRNAs will be produced, and different proteins will be expressed. The idea is that a K+ channel gene, in this case the gene for Shaker, can produce several different subunits through alternative splicing. 10 Figure 10. Schematic showing how alternative splicing of the RNA transcribed from a single gene can produce a variety of different proteins. In A, a gene is transcribed to produce an RNA with 4 exons and 3 introns. Following transcription, the introns are cut out and the all four exons are spliced together. The mRNA composed of the 4 exons is then translated into a polypeptide. In B, the same gene is transcribed but two different mRNAs are formed due to alternative splicing. In the top, exons 1, 3 and 4 are spliced together and that mRNA is translated into polypeptide 1, having an amino acid sequence different from the mRNA in A, which contained all 4 exons. Yet a different splice variant, and hence a different polypeptide, is formed by splicing exons 1,2 and 4 together. A second factor generating K+ channel diversity is that the K+ channel can be constructed from four of the same subunit type, called a homomeric channel, or the channel can be formed by combing two or more different types of subunits. Channels composed of different subunit types are called heteromeric channels. Each combination of subunit types produces a K+ channel with unique functional properties. Figure 11. Homomeric K+ channels are composed of 4 idential subunits. Heteromeric K+ channels are formed by different types of subunits. The different subunit types are due to alternative splicing of the K+ gene. Channels composed of different subunits have different electrical properties. 11 The pore and the selectivity filter The operation of the pore and how the selectivity filter in the pore endows the channel with its ability to pass only one type of ion, K+ in this case, was shown by Rod MacKinnon and his colleagues. This was a stunning achievement and MacKinnon was awarded the Nobel Prize in Chemistry in 2003. They did this by actually seeing the movement of K+ ions in the channel with Xray crystallography. To obtain enough channels to form a crystal, they used a bacterial K+ channel called KcsA from the bacterium, Steptomyces lividans. It is important to note that the KcsA channel has a simple topology with only two membrane spanning segments per subunit. The two segments correspond to the Shaker K+ channel without S1 through S4 (the KcsA channel has no voltage sensor and is gated by acidity rather than by voltage). Four subunits, each with only two membrane spanning segments, comprise the channel (Fig. 12). Each subunit, in turn, has two protein helices, an outer helix that faces the lipid membrane and an inner helix that faces the central pore (Fig. 12). The inner and outer helices are separated by a series of amino acids that form the pore helix and the selectivity filter. Figure 12. Each subunit of the KcsA K+ channel is composed an outer and inner helix connected by a group of amino acids that form the pore region. The pore region has two major components: 1) the pore helix; and 2) the selectivity filter (not shown). Lower left panel: Top view of a K+ channel composed of four subunits, where each subunit is shown as a different color. The inner helices of the blue and red subunits are indicated by arrows while the outer helices of the green and yellow subunits are indicated by arrows. Lower right: The inner helices of four subunits are arranged as an inverted teepee. The four pore helices are shown in white. The sequence of amino acids in the selectivity filter, the sequence important for K+ selectivity, is the same in the pores of all K+ potassium channels. The entire sequence is composed of four pore loops inside the pore, one loop from each of the four subunits. MacKinnon called these amino acids the K+ signature sequence (Fig. 13). Mackinnon was profoundly struck by this universal feature of K+ channels and in his 2003 Nobel Address he wrote, "The conservation of the signature sequence amino acids in K+ channels throughout the tree of life, from bacteria to higher eukaryotic cells, imp...
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- Spring '08