Single channel function of recombinant type-1 inositol 1,4,5-trisphosphatereceptor ligand binding domain splice variants

In this study we describe the expression and function of the two rat type-1 inositol 1,4,5-trisphosphate receptor (InsP(3)R) ligand binding domain spl ice variants (SI +/- SII+). Receptor protein from COS-1 cells transfected w ith the type-1 InsP(3)R expression plasmids (plnsP(3)R-T1, plnsP(3)R-T1ALT) or control DNA were incorporated into planar lipid bilayers and the single channel properties of the recombinant receptors were defined. The unitary conductance of the two splice variants were similar to 290 pS with Cs+ as c harge carrier and similar to 65 pS with Ca2+ as charge carrier. Both InsP(3 )R expression products consistently behaved like those of the native type-1 receptor isoform isolated from cerebellum in terms of their InsP(3), Ca2+, and heparin sensitivity. An InsP(3) receptor ligand binding domain truncat ion lacking the 310 amino-terminal amino acids (plnsP(3)R-Delta T1ALT) form ed tetrameric complexes but failed to bind InsP(3) with high affinity, and did not form functional Ca2+ channels when reconstituted in lipid bilayers. These data suggest that 1) the ligand binding alternative splice site is f unctionally inert in terms of InsP(3) binding and single channel function, and 2) the single channel properties of the expressed recombinant type-1 ch annel are essentially identical to those of the native channel. This work e stablishes a foundation from which molecular/ biophysical approaches can be used to define the structure-function properties of the InsP(3) receptor c hannel family.