NDLI: A novel crystallization method for visualizing the membrane localization of potassium channels.

Content Provider	PubMed Central
Author	Lopatin, A. N. Makhina, E. N. Nichols, C. G.
Abstract	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.
Related Links	http://dx.doi.org/10.1016/s0006-3495(98)77925-1
Ending Page	2170
Page Count	12
Starting Page	2159
File Format	PDF
ISSN	00063495
e-ISSN	15420086
Journal	Biophysical Journal
Issue Number	5
Volume Number	74
Language	English
Publisher Date	1998-05-01
Access Restriction	Open
Subject Keyword	Biophysics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biophysics

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