期刊
BIOPHYSICAL JOURNAL
卷 79, 期 3, 页码 1388-1399出版社
BIOPHYSICAL SOCIETY
DOI: 10.1016/S0006-3495(00)76391-0
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资金
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL058851] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF MENTAL HEALTH [R29MH053367] Funding Source: NIH RePORTER
- NHLBI NIH HHS [R01HL58851, R01HL570832] Funding Source: Medline
- NIMH NIH HHS [R29MH53367] Funding Source: Medline
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 Ca2+-sensitive ion channels. The unitary conductance (similar to 250 pS; 220/20 mM Cs+ as charge carrier), gating, InsP(3), and Ca2+ 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 lull-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.
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