Shaker channel

Shaker-channels, eag (ether- -go-go)-channels, slo (slow-poke)-channels were cloned from behavioral Drosophila melanogaster mutants. The channels were named according to the Drosophila mutant phenotype, Shaker, ether-go-go, slow-poke. Subsequently, eag-cDNA was used to clone related voltage-gated potassium channel subunits erg (eag-related) and elk (eag-like). The human erg ortholog (HERG) mediates cardiac DCS. 

In addition to the membrane-inserted core domain of Kv channels, their cytoplasmic domains have important roles for Kv-channel function [5]. Many of these functions are related to subunits assembly, channel trafficking to and from the plasma membrane, and interactions with cytoskeletal components (Fig. la). A tetramerization (T) domain for subunit assembly has been well defined in Shaker-channels, where it is localized in the amino-terminus. Other Kv-channels (e.g., eag, HERG, KvLQTl) may have comparable domains within the cytoplasmic carboxy-terminus. ER retention and retrieval signals have been found... 

Properties of Shaker channels in Drosophila muscle and photoreceptor cells ... 

Fig. 3. (A) Model of the proposed pore forming part of K channel subunits. Segments S5 and S6 are possibly membrane-spanning helices. The helices are connected by a hydrophobic segment H5 which may be tucked into the lipid bilayer [48]. H5 is flanked by two proline residues P. Adjacent to these proline residues are amino acid side chains ( ) important for external TEA binding [45,46]. Approximately halfway between these two proline residues are amino acid side chains ( ) affecting internal TEA binding [46,47] and K channel selectivity [48]. (B) Mutations are indicated which affect in Shaker channels external TEA (TEAe) or internal TEA (TEA,) binding. Concentrations of TEA for half block of the wild-type and mutant K channels are given at the right-hand side of the corresponding sequence. Data have been compiled from [45-47].

Fig. 4. Schematic organization of the Shaker K channel gene. The coordinates of the physical map are as in [9]. The direction of transcription is indicated by arrows. Approximate location of exons is given by boxes. Open box corresponds to noncoding exons, lettered boxes to alternative amino-terminal ends of Shaker channel proteins and the core region, respectively, numbered boxes to the two alternative carboxy-terminal ends. Exon numbers are as in [53].

A mutant Shaker channel lacking 42 amino acids near the amino terminus opened in response to depolarization but did not inactivate (see Figure 13.28). Most revealing, inactivation was restored by adding a synthetic peptide corresponding to the first 20 residues of the native channel. 

Studies with mutant Shaker channels support this model for operation of the S4 helix in voltage sensing. When one or more arginine or lysine residues in the S4 helix of the Shaker channel were replaced with neutral or acidic residues, fewer positive charges than normal moved across the membrane in response to a membrane depolarization, indicating that arginine and lysine residues in the S4 helix do indeed move across the membrane. In other studies, mutant... 

A EXPERIMENTAL FIGURE 7-38 Experiments with a mutant channel lacking the N-terminai giobular domains support the ball-and-chain inactivation modei. The wild-type Shaker channel and a mutant form lacking the amino acids composing the N-terminal ball were expressed in Xenopus oocytes. The activity of the channels then was monitored by the patch-clamp technique. When patches were depolarized from -0 to -1-30 mV, the wild-type channel opened for =5 ms and then closed (red curve), whereas the mutant channel opened normally, but could not close (green curve). When a chemically synthesized ball peptide was added to the cytosolic face of the patch, the mutant channel opened normally and then closed (blue curve). This demonstrated that the added peptide inactivated the channel after it opened and that the ball does not have to be tethered to the protein in order to function. [From W. N. Zagotta et al., 1990, Science 250 568.]... 

Match the sodium (eel electric organ) and potassium (Shaker) channels "with the corresponding properties listed in the right column. 

There are two pieces of experimental evidence given in the text in support of the ball-and-chain model of channel inactivation. The first is that treatment of the cytoplasmic side of either the Na" or K" channel with trypsin yields a trimmed channel that stays open after depolarization. The second is that N-terminal splice variants of the potassium channel have altered inactivation kinetics. A deletion of 42 amino acids at the N-terminus of the Shaker channel causes the channel to open upon depolarization but not inactivate. Addition of a synthetic peptide corresponding to the deleted amino acids restores inactivation to the channel. 

The electrode resistance values were measured to confirm that the electrodes were electrically isolated and that the apertures were open. Figure 6 shows a macro-patch recording obtained when a devitellinized oocyte, expressing Shaker potassium channels, was dropped onto an aperture. The ionic currents carried by a population of 80 Shaker channels were measured in response... 

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