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Biophys J, July 2001, p. 117-124, Vol. 81, No. 1

Single-Channel Recordings of Recombinant Inositol Trisphosphate Receptors in Mammalian Nuclear Envelope

Darren Boehning,* Suresh K. Joseph,* Don-On Daniel Mak,dagger and J. Kevin Foskettdagger

 *Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19103 USA; and  dagger Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 USA

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are intracellular Ca2+ channels gated by the second messenger InsP3. Here we describe a novel approach for recording single-channel currents through recombinant InsP3Rs in mammalian cells that applies patch-clamp electrophysiology to nuclei isolated from COS-7 cells transiently transfected with the neuronal (SII(+)) and peripheral (SII(-)) alternatively-spliced variants of the rat type 1 InsP3R. Single channels that were activated by InsP3 and inhibited by heparin were observed in 45% of patches from nuclei prepared from transfected cells overexpressing recombinant InsP3Rs. In contrast, nuclei from cells transfected with the vector alone had InsP3-dependent channel activity in only 1.5% of patches. With K+ (140 mM) as the permeant ion, recombinant SII(+) and SII(-) channels had slope conductances of 370 pS and 390 pS, respectively. The recombinant channels were 4-fold more selective for Ca2+ over K+, and their open probabilities were biphasically regulated by cytoplasmic [Ca2+]. This approach provides a powerful new methodology to study the permeation and gating properties of recombinant mammalian InsP3Rs in a native mammalian membrane environment at the single-channel level.

Biophys J, July 2001, p. 117-124, Vol. 81, No. 1
© 2001 by the Biophysical Society   0006-3495/01/07/117/08  $2.00


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