Inositol 1,4,5-Trisphosphate and Calcium Regulate the Calcium Channel Function of the Hepatic Inositol 1,4,5-Trisphosphate Receptor

doi:10.1074/jbc.272.5.2675

Inositol 1,4,5-Trisphosphate and Calcium Regulate the Calcium Channel Function of the Hepatic Inositol 1,4,5-Trisphosphate Receptor*

From the Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

^‡ Recipient of the Advanced Research Training Award from the American Gastroenterology Association. To whom correspondence should be addressed:
Dept. of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111.
Tel.: 617-636-6739; Fax: 617-636-0445; E-mail: JDUFOUR{at}OPAL.TUFTS.EDU.

Abstract

The regulation of the inositol 1,4,5-trisphosphate (IP₃) receptor in liver was analyzed using a novel superfusion method. Hepatic microsomes were loaded with ⁴⁵Ca²⁺, and superfused at high flow rates to provide precise control over IP₃ and Ca²⁺ concentrations ([Ca²⁺]) and to isolate ⁴⁵Ca²⁺ release from reuptake. ⁴⁵Ca²⁺ release was dependent on both [Ca²⁺] and IP₃. The initial rate of ⁴⁵Ca²⁺ release was a biphasic function of [Ca²⁺], increasing as [Ca²⁺] approached 3 μM but decreasing at higher concentrations, suggesting that the hepatic IP₃ receptor is regulated by [Ca²⁺] at two sites, a high affinity potentiation site and a low affinity inhibitory site. The relationship between initial rates and IP₃ concentration was steep (Hill coefficient of 3.4), suggesting that activation of the calcium channel requires binding of at least 3 IP₃ molecules. IP₃ concentrations above 10 μM produced rapid decay of release rates, suggesting receptor inactivation. Superfusion with 10 μM IP₃ under conditions that minimize calcium release ([Ca²⁺] < 1 nM) inhibited ⁴⁵Ca²⁺ release in response to subsequent stimulation (400 nM Ca²⁺). These data suggest sequential positive and negative regulation of the hepatic IP₃ receptor by cytosolic calcium and by IP₃, which may underlie hepatocellular propagation of regenerative, oscillatory calcium signals.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants DK35652 and DK34928 (to I. M. A.) and NS28815 (to T. J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

IP₃

D-myo-inositol 1,4,5-trisphosphate

DTT

2,3-dihydroxybutane-1,4-dithiol

MOPS

3-[N-morpholino]propansulfonic acid.
- Received September 17, 1996.
- Revision received November 8, 1996.