Single-channel function of recombinant type 2 inositol 1,4, 5-trisphosphate receptor.
A full-length rat type 2 inositol 1,4,5-trisphosphate (InsP(3)) receptor cDNA construct was generated and expressed in COS-1 cells. Targeting of the full-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purified antibodies and InsP(3)R-green fluorescent protein chimeras. The receptor protein was solubilized and incorporated into proteoliposomes for functional characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed InsP(3)- and Ca(2+)-sensitive ion channels. The unitary conductance ( approximately 250 pS; 220/20 mM Cs(+) as charge carrier), gating, InsP(3), and Ca(2+) sensitivities were similar to those previously described for the native type 2 InsP(3)R channel. However, the maximum open probability of the recombinant channel was slightly lower than that of its native counterpart. These data show that our full-length rat type 2 InsP(3)R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the native type 2 InsP(3)R channel. The possibility of measuring the function of single recombinant type 2 InsP(3)R is a significant step toward the use of molecular tools to define the determinants of isoform-specific InsP(3)R function and regulation.
Ramos-Franco
J
J
Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153 USA.
Bare
D
D
Caenepeel
S
S
Nani
A
A
Fill
M
M
Mignery
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D015220:Calcium Channels / Q000737:chemistry / Q000502:physiology*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000502:physiology; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011510:Proteolipids; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008051:Lipid Bilayers; D011510:Proteolipids; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; C020485:proteoliposomes; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium