M Li1, N Unwin, K A Stauffer, Y N Jan, L Y Jan. 1. Howard Hughes Medical Institute, Department of Physiology, University of California, San Francisco 94143-0724.
Abstract
BACKGROUND: Voltage-gated K+ channels play an important role in the control of neuronal excitability and synaptic plasticity. Their low abundance and extraordinary heterogeneity have rendered their purification from natural sources difficult. We have previously cloned a voltage-gated K(+)-channel gene, Shaker, from Drosophila. The Shaker K(+)-channel polypeptide resembles one of the four internal repeats of a Na(+)- or Ca(2+)-channel alpha subunit, suggesting that this example of a K+ channel contains four identical or homologous subunits. Similar K(+)-channel polypeptides have been characterized from mammals, other vertebrate and invertebrate species, and from plants. Electrophysiological studies of K+ channels expressed in Xenopus oocytes suggest that they are indeed tetramers, and heteromultimeric K+ channels have been found in the mammalian brain. Until now, however, no K+ channel, nor any other member of the superfamily of voltage-gated ion channels, has been characterized by electron microscopy or other structural analysis. RESULTS: We have purified Shaker K+ channels, expressed in insect Sf9 cells, to apparent homogeneity, and imaged them using the electron microscope. The physical dimensions of these molecules, as well as their biochemical characteristics, are consistent with a tetrameric subunit composition. Moreover, the Shaker channel revealed by negative staining has the appearance of a four-fold symmetric tetramer, with a large, central vestibule that presumably constitutes part of the pathway for ions. CONCLUSION: These first clear images of a voltage-gated ion channel reveal a marked four-fold symmetry. The integrity of the purified tetrameric complex indicates that the purification scheme used in this study may be further developed for future structural analysis of voltage-gated K+ channels.
BACKGROUND: Voltage-gated K+ channels play an important role in the control of neuronal excitability and synaptic plasticity. Their low abundance and extraordinary heterogeneity have rendered their purification from natural sources difficult. We have previously cloned a voltage-gated K(+)-channel gene, Shaker, from Drosophila. The Shaker K(+)-channel polypeptide resembles one of the four internal repeats of a Na(+)- or Ca(2+)-channel alpha subunit, suggesting that this example of a K+ channel contains four identical or homologous subunits. Similar K(+)-channel polypeptides have been characterized from mammals, other vertebrate and invertebrate species, and from plants. Electrophysiological studies of K+ channels expressed in Xenopus oocytes suggest that they are indeed tetramers, and heteromultimeric K+ channels have been found in the mammalian brain. Until now, however, no K+ channel, nor any other member of the superfamily of voltage-gated ion channels, has been characterized by electron microscopy or other structural analysis. RESULTS: We have purified Shaker K+ channels, expressed in insect Sf9 cells, to apparent homogeneity, and imaged them using the electron microscope. The physical dimensions of these molecules, as well as their biochemical characteristics, are consistent with a tetrameric subunit composition. Moreover, the Shaker channel revealed by negative staining has the appearance of a four-fold symmetric tetramer, with a large, central vestibule that presumably constitutes part of the pathway for ions. CONCLUSION: These first clear images of a voltage-gated ion channel reveal a marked four-fold symmetry. The integrity of the purified tetrameric complex indicates that the purification scheme used in this study may be further developed for future structural analysis of voltage-gated K+ channels.