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 localiza tion of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1 ), were expressed in Xenopus oocytes and loaded with Br-ions by microi njection. Oocytes were then exposed to extracellular thallium. Under c onditions favoring influx of Tl+ ions (negative membrane potential und er voltage clamp, or high concentration of extracellular Tl+), crystal s of TlBr, visible under low-power microscopy, formed under the membra ne 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 mu S K+ conductance. The number of observed crystals was much lo wer than the estimated number of functional channels. Based on the pat tern of crystal formation, K+ channels appear to be expressed mostly a round the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distributi on 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 oocy tes under two-microelectrode voltage clamp. This technique, together w ith direct patch-clamping of patches of membrane in regions of high cr ystal density, confirmed that the localization of TIBr crystals corres ponded to the localization of functional K+ channels and suggested a c lustered organization of functional channels. With appropriate permean t ion/counterion pairs, this approach may be applicable to the visuali zation of the membrane distribution of any functional ion channel.