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 TIBr, 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 μS 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.