IMAGES OF PURIFIED SHAKER POTASSIUM CHANNELS

Background: Voltage-gated K+ channels play an important role in the co ntrol of neuronal excitability and synaptic plasticity. Their low abun dance and extraordinary heterogeneity have rendered their purification from natural sources difficult. We have previously cloned a voltage-g ated K+-channel gene, Shaker, from Drosophila, The Shaker K+-channel p olypeptide resembles one of the four internal repeats of a Na+- or Ca2 +-channel a subunit, suggesting that this example of a K+ channel cont ains four identical or homologous subunits. Similar K+-channel polypep tides have been characterized from mammals, other vertebrate and inver tebrate species, and from plants. Electrophysiological studies of K+ c hannels expressed in Xenopus oocytes suggest that they are indeed tetr amers, and heteromultimeric K+ channels have been found in the mammali an brain. Until now, however, no K+ channel, nor any other member of t he superfamily of voltage-sated ion channels, has been characterized b y electron microscopy or other structural analysis. Results: We have p urified Shaker KC channels, expressed in insect Sf9 cells, to apparent homogeneity, and imaged them using the electron microscope. The physi cal dimensions of these molecules, as well as their biochemical charac teristics, are consistent. with a tetrameric subunit composition. More over, the Shaker channel revealed by negative staining has the appeara nce 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 mark ed four-fold symmetry. The integrity of the purified tetrameric comple x indicates that the purification scheme used in this study may be fur ther developed for future structural analysis of voltage-gated K+ chan nels.