Single-channel recordings of recombinant inositol trisphosphate receptors in mammalian nuclear envelope.
Inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) are intracellular Ca(2+) channels gated by the second messenger InsP(3). Here we describe a novel approach for recording single-channel currents through recombinant InsP(3)Rs 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 InsP(3)R. Single channels that were activated by InsP(3) and inhibited by heparin were observed in 45% of patches from nuclei prepared from transfected cells overexpressing recombinant InsP(3)Rs. In contrast, nuclei from cells transfected with the vector alone had InsP(3)-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 Ca(2+) over K(+), and their open probabilities were biphasically regulated by cytoplasmic [Ca(2+)]. This approach provides a powerful new methodology to study the permeation and gating properties of recombinant mammalian InsP(3)Rs in a native mammalian membrane environment at the single-channel level.
Boehning
D
D
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19103, USA.
Joseph
S K
SK
Mak
D O
DO
Foskett
J K
JK
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004594:Electrophysiology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015640:Ion Channel Gating / Q000187:drug effects; D009685:Nuclear Envelope / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011336:Probability; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D002118:Calcium