A novel crystallization method for visualizing the membrane localization of potassium channels.
The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.
Lopatin
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AN
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. anatoli@cellbio.wustl.edu
Makhina
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EN
Nichols
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CG
D000818:Animals; D001703:Biophysics / Q000379:methods; D001965:Bromides; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure*; D003460:Crystallization; D051668:Delayed Rectifier Potassium Channels; D005260:Female; D008962:Models, Theoretical; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000032:analysis* / Q000096:biosynthesis; D024661:Potassium Channels, Inwardly Rectifying; D024642:Potassium Channels, Voltage-Gated; D017680:Potassium Compounds; D012680:Sensitivity and Specificity; D051669:Shab Potassium Channels; D014982:Xenopus laevis
D001965:Bromides; D051668:Delayed Rectifier Potassium Channels; C099489:KCNJ4 protein, human; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D024642:Potassium Channels, Voltage-Gated; D017680:Potassium Compounds; D051669:Shab Potassium Channels; C039004:potassium bromide