Biophys J, December 1998, p. 2783-2793, Vol. 75, No. 6

Single Channel Function of Recombinant Type-1 Inositol 1,4,5-Trisphosphate Receptor Ligand Binding Domain Splice Variants

 ^*Department of Physiology and  ^#Cardiovascular Institute, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153 USA

In this study we describe the expression and function of the two rat type-1 inositol 1,4,5-trisphosphate receptor (InsP₃R) ligand binding domain splice variants (SI±/SII+). Receptor protein from COS-1 cells transfected with the type-1 InsP₃R expression plasmids (pInsP₃R-T1, pInsP₃R-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 ~290 pS with Cs⁺ as charge carrier and ~65 pS with Ca²⁺ as charge carrier. Both InsP₃R expression products consistently behaved like those of the native type-1 receptor isoform isolated from cerebellum in terms of their InsP₃, Ca²⁺, and heparin sensitivity. An InsP₃ receptor ligand binding domain truncation lacking the 310 amino-terminal amino acids (pInsP₃R-T1ALT) formed tetrameric complexes but failed to bind InsP₃ 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 functionally inert in terms of InsP₃ binding and single channel function, and 2) the single channel properties of the expressed recombinant type-1 channel are essentially identical to those of the native channel. This work establishes a foundation from which molecular/biophysical approaches can be used to define the structure-function properties of the InsP₃ receptor channel family.

Biophys J, December 1998, p. 2783-2793, Vol. 75, No. 6
© 1998 by the Biophysical Society   0006-3495/98/12/2783/11  $2.00

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