Calcium release-activated calcium current in rat hepatocytes

G.Y. Rychkov¹, T. Litjens¹, M.L. Roberts¹ and G.J. Barritt², ¹School of Molecular and Biomedical Sciences, University of Adelaide, SA 5005 and ²Flinders Medical Centre, Flinders University of South Australia, SA 5001 Australia.

Store-operated Ca²⁺ (SOC) channels play a central role in regulating intracellular Ca²⁺ concentration in hepatocytes and other nonexcitable animal cells (Gregory & Barritt, 2003). A major function of SOC channels appears to be to replenish intracellular Ca²⁺ stores when intracellular Ca²⁺ is lost from the cell during agonist-induced increases in the cytoplasmic Ca²⁺ concentration. One of the best-known store operated channels, Ca²⁺release-activated Ca²⁺ (CRAC) channel has been extensively characterised in a number of immortalised cell lines. There is little evidence, however, that I_CRAC is activated in the physiological conditions in cells in primary culture. It has been speculated that the highly Ca²⁺-specific CRAC channels are only expressed in blood cells and in transformed cells.

The aim of the present experiments was to elucidate the properties of the SOC channels in rat hepatocytes. Hepatocytes were isolated by collagenase digestion and plated on glass coverslips. Patch-clamp recording was conducted in the whole-cell mode using standard procedures after 24-48 hours.

Depletion of intracellular Ca²⁺ stores in rat hepatocytes activated a Ca²⁺-selective inward current. Properties of this current, including high selectivity for Ca²⁺, strong inward rectification, fast Ca²⁺ dependent inactivation at negative potentials and block by La³⁺ and 2-APB, were similar or identical to those of I_CRAC found in mast cells, RBL cells, Jurkat T lymphocytes (Zweifach & Lewis, 1993; Hoth & Penner, 1992; Bakowski & Parekh, 2002) and H4-IIE liver cells (Rychkov et al. 2001). The amplitude of I_CRAC in rat hepatocytes varied between −30 and −120 pA at −100 mV with an average density of about −1 pA/pF. Extracellular application of vasopressin or ATP activated a current with the same properties and the same size as that observed by InsP₃ induced depletion of the stores. I_CRAC developed more slowly with vasopressin than with ATP as agonist. Increasing the concentration of ATP shortened the delay in the development of I_CRAC, but did not change the amplitude of the current or the rate of its development. Concentrations of ATP (5-10 μM) that cause waves of increased cytoplasmic Ca²⁺ concentration also activated I_CRAC. So far, no other type of current activated by Ca²⁺ store depletion has been detected in these cells. It is concluded that CRAC channels are the major, and possibly the only, type of SOC channel in rat hepatocytes.

Bakowski, D. & Parekh, A.B. (2002) Cell Calcium 32, 379-391.

Gregory, R.B. & Barritt, G.J. (2003) Biochemical Journal 369, 1-7.

Hoth, M. & Penner, R. (1992) Nature 355, 353-356.

Rychkov, G., Brereton, H.M., Harland, M.L. & Barritt, G.J. (2001) Hepatology 33, 938-947.

Zweifach, A. & Lewis, R.S. (1993) Proceedings of the National Academy of Sciences of the United States of America 90, 6295-6299.