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 ICRAC 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 ICRAC 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 ICRAC 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. ICRAC developed more slowly with vasopressin than with ATP as agonist. Increasing the concentration of ATP shortened the delay in the development of ICRAC, 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 ICRAC. 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.