APPS November 2002 Meeting Abstract 2415

Grigori Y. Rychkov, M. Lyn Harland, Gregory J. Barritt, Department of Physiology, University of Adelaide, and Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide.

In patch-clamp recording, during hyperpolarizing pulses to potentials more negative than -80 mV, Ca²⁺ release activated Ca²⁺ (CRAC) channels rapidly inactivate with a double exponential time course. This inactivation is unaffected by increasing the concentration of EGTA in the pipette solution but is significantly reduced when a faster Ca²⁺ buffer, BAPTA, is used instead of EGTA, or when Ba²⁺is used instead of Ca²⁺ as the charge carrier¹. This suggests that fast inactivation of CRAC channels is mediated by the binding of Ca²⁺ close to the internal mouth of the channel. The exact mechanism of this process, however, remains unknown. Ca²⁺dependent inactivation is a well-known phenomenon for different types of voltage-dependent Ca²⁺ channels². It has been shown recently that over expression of a mutant form of calmodulin lacking functional EF hands eliminates Ca²⁺ dependent inactivation of L-type and P/Q-type voltage-dependent Ca²⁺ channels³.

In the present work we investigated the possibility that a calmodulin-dependent mechanism similar to that which regulates voltage-dependent Ca²⁺channels is also responsible for the fast inactivation of CRAC channels in H4IIE cells, an immortalised rat liver cell line. We show here that over-expression of the calmodulin binding domain of type I adenylyl cyclase significantly alters the fast inactivation of I_CRAC. The relative amplitude of the fast inactivating component of I_CRAC was decreased while the non-inactivating component increased. This suggests that calmodulin is involved in the Ca²⁺dependent gating of CRAC channels.