Tom Litjens’s research while affiliated with University of Adelaide and other places

Physiologically, hormone induced release of Ca²+ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP₃) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP₃ receptors act as channels, releasing Ca²+ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca²+ stores leads to activation of store-operated Ca²+ channels on the plasma membrane which replenishes lost Ca²+ and sustain Ca²+ signalling. There are three isoforms of IP₃ receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca²+ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca²+ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca²+ channels, and Ca²+ release activated Ca²+ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP₃ receptor but not the others. Here we show that H4IIE liver cells express all three types of IP₃ receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I(CRAC)), while type 1 and type 2, but not type 3, participate in observed Ca²+ release in response to receptor stimulation. Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca²+ store linked to the activation of I(CRAC) predominantly expresses type 1 IP₃ receptors.

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca2+ release activated Ca2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca2+ sensor required for activation of CRAC current (I(CRAC)) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I(CRAC). In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I(CRAC). The results show that cells expressing low Orai1 : STIM1 ratios produce I(CRAC) with strong fast Ca2+-dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I(CRAC) with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca2+, Ba2+ and Sr2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

Oscillatory increases in the cytoplasmic Ca ²⁺ concentration ([Ca ²⁺ ] cyt ) play essential roles in the hormonal regulation of liver cells. Increases in [Ca ²⁺ ] cyt require Ca ²⁺ release from the endoplasmic reticulum (ER) and Ca ²⁺ entry across the plasma membrane. Store‐operated Ca ²⁺ channels (SOCs), activated by a decrease in Ca ²⁺ in the ER lumen, are responsible for maintaining adequate ER Ca ²⁺ . Experiments using patch‐clamp recording and the fluorescent Ca ²⁺ reporter fura‐2 indicate there is only one type of SOC in rat liver cells. These SOCs have a high selectivity for Ca ²⁺ and properties essentially indistinguishable from those of Ca ²⁺ release‐activated Ca ²⁺ (CRAC) channels. Although Orai1, a CRAC channel pore protein, and stromal interaction molecule 1 (STIM1), a CRAC channel Ca ²⁺ sensor, are components of liver cell SOCs, the mechanism of activation of SOCs, and in particular the role of subregions of the ER, are not well understood. Recent experiments have used the transient receptor potential vanilloid 1 (TRPV1) non‐selective cation channel, ectopically expressed in liver cells, and a choleretic bile acid to deplete Ca ²⁺ from different ER subregions. The results of these studies have provided evidence that only a small component of the ER is required for STIM1 redistribution and the activation of SOCs. It is concluded that different Ca ²⁺ microdomains in the ER and cytoplasmic space are important in both the activation of SOCs and in the signalling actions of Ca ²⁺ in liver cells. Future experiments will investigate the nature of these microdomains further.

Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-gamma (phospholipase C-gamma) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-gamma-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-gamma has not been defined clearly. The aim of the present study was to determine if PLC-gamma is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-gamma1 caused a reduction (by approx. 70%) in the PLC-gamma1 protein expression, with maximal effect at 72-96 h. This was associated with a decrease (by approx. 60%) in the amplitude of the I(SOC) (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP(3) or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the I(SOC) amplitude. Immunoprecipitation of PLC-gamma1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-gamma1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.

Hepatocytes are highly polarised epithelial cells that mediate a large number of metabolic pathways, the transcellular movement of numerous ions and metabolites, and the secretion of proteins from both basal and canalicular membrane regions. Hormone-induced changes in the concentration of intracellular Ca2+ play a central role in regulating these functions. Store-operated Ca2+ channels (SOCs) and other Ca2+-permeable channels in the plasma membrane which are activated by hormones are essential for regulating the amount of Ca2+ in the hepatocyte in order to allow these Ca2+ signalling processes to occur. However, the properties of hormone-activated Ca2+ channels in hepatocytes and in other epithelial cells are not well defined. In this study, we have investigated SOCs in cultured rat hepatocytes by patch-clamp recording using IP3 and hormones as activators. We show that IP3 activates a single type of SOC, which, on the basis of its high selectivity for Ca2+ over Na+, inhibition by La3+ and 2-aminoethyl diphenylborate (2-APB), and the time course of fast inactivation, is very similar to CRAC channel in mast cells and lymphocytes. Moreover, a current (ISOC) with properties identical to those of the IP3-activated current can be activated by physiological concentrations of ATP and vasopressin. It is concluded that SOCs with properties similar to those of CRAC channel are present in hepatocytes, highly differentiated primary cells, and these channels can be activated by hormones under conditions close to physiological.

Vasopressin and other phospholipase-C-coupled hormones induce oscillations (waves) of [Ca2+]cyt (cytoplasmic Ca2+ concentration) in liver cells. Maintenance of these oscillations requires replenishment of Ca2+ in intracellular stores through Ca2+ inflow across the plasma membrane. While this may be achieved by SOCs (store-operated Ca2+ channels), some studies in other cell types indicate that it is dependent on AA (arachidonic acid)-activated Ca2+ channels. We studied the effects of AA on membrane conductance of rat liver cells using whole-cell patch clamping. We found no evidence that concentrations of AA in the physiological range could activate Ca2+-permeable channels in either H4IIE liver cells or rat hepatocytes. However, AA (1-10 microM) did inhibit (IC50=2.4+/-0.1 microM) Ca2+ inflow through SOCs (ISOC) initiated by intracellular application of Ins(1,4,5)P3 in H4IIE cells. Pre-incubation with AA did not inhibit ISOC development, but decreased maximal amplitude of the current. Iso-tetrandrine, widely used to inhibit receptor-activation of phospholipase A2, and therefore AA release, inhibited ISOC directly in H4IIE cells. It is concluded that (i) in rat liver cells, AA does not activate an AA-regulated Ca2+-permeable channel, but does inhibit SOCs, and (ii) iso-tetrandrine and tetrandrine are effective blockers of CRAC (Ca2+-release-activated Ca2+) channel-like SOCs. These results indicate that AA-activated Ca2+-permeable channels do not contribute to hormone-induced increases or oscillations in [Ca2+]cyt in liver cells. However, AA may be a physiological modulator of Ca2+ inflow in these cells.

LAMP-1 (lysosome-associated membrane protein), a major glycoprotein present in the lysosomal membrane, constitutes up to 50% of total membrane proteins. LAMP-1, expressed at the plasma membrane, is reported to be the major molecule expressing the sialyl-Lewis X antigen. Two forms of LAMP-1 exist; the full-length LAMP-1 [LAMP-1 (+Tail)] has a highly glycosylated lumenal domain, a membrane-spanning domain and a short cytoplasmic tail, and the truncated LAMP-1 [LAMP-1 (-Tail)] contains only the lumenal domain. Soluble LAMP-1 (+/-Tail) has been reported in circulation. LAMP-1 at the cell surface has been shown to interact with E-selectin and galectin and is proposed to function in cell-cell interactions. However, the functional role(s) of soluble LAMP-1 in circulation is unclear. To investigate the functional role of soluble LAMP-1 in circulation, recombinant LAMP-1 (-Tail) and LAMP-1 (+Tail) were produced in HT1080 cells. Two immune-quantification assays were developed to distinguish between the LAMP-1 forms. The interaction and aggregation properties of the different LAMP-1 forms were investigated using the immune-quantification assays. Only LAMP-1 (+Tail) was found to aggregate and interact with plasma proteins. Plasma proteins that interact with LAMP-1 were isolated by affinity chromatography with either the recombinant LAMP-1 (-Tail) or a synthesized peptide consisting of the 14 amino acids of the LAMP-1 cytoplasmic tail. Transthyretin was found to interact with the cytoplasmic tail of LAMP-1. Transthyretin exists as a homotetramer in plasma, as such may play a role in the aggregation of LAMP-1 in circulation.

Store-operated Ca2+ channels (SOCs) provide a major pathway for Ca2+ entry in non-excitable cells. SOCs in immortalized liver cells are highly selective for Ca2+ over other cations and are similar to well-studied Ca2+ release activated Ca2+ (CRAC) channels in haematopoietic cell lines. In the present work, employing H4IIE liver cells, we investigated fast inactivation of SOC current (ISOC), which occurs at membrane potentials below -60 mV. This inactivation was significantly reduced when BAPTA, a faster Ca2+ buffer, was used instead of EGTA, and was completely abolished if Na+ was used as a charge carrier in the absence of divalent cations in the external medium. These results suggested that fast inactivation of SOCs in H4IIE cells was Ca2+ dependent and was similar to the fast inactivation of CRAC channels. Experiments showing that the fast inactivation of ISOC was not affected by the disruption of actin by latrunculin B indicate that the cytoskeleton is unlikely to be involved. To elucidate the mechanism of Ca2+ dependence, a possible role of calmodulin (CaM) in SOCs' fast inactivation was investigated. The CaM inhibitors Mas-7 and calmidazolium failed to affect ISOC fast inactivation, whereas over-expression of a CaM inhibitor peptide or a mutant CaM lacking functional EF hands significantly altered the inactivation of ISOC. Out of two exponential components normally required to approximate kinetics of ISOC fast inactivation, the faster component was reduced in amplitude by 30%, compared to the control. The results presented suggest that CaM is responsible for at least part of Ca(2+)-dependent fast inactivation of ISOC in liver cells. It is hypothesized that CaM is tethered to the channel itself and therefore protected from chemical inhibitors.

The lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (4-sulfatase) is required for the degradation of the glycosaminoglycan substrates dermatan and chondroitin sulfate. A 4-sulfatase deficiency results in the accumulation of undegraded substrate and causes the severe lysosomal storage disorder mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome. A wide variation in clinical severity is observed between MPS VI patients and reflects the number of different 4-sulfatase mutations that can cause the disorder. The most common 4-sulfatase mutation, Y210C, was detected in approximately 10% of MPS VI patients and has been associated with an attenuated clinical phenotype when compared to the archetypical form of MPS VI. To define the molecular defect caused by this mutation, Y210C 4-sulfatase was expressed in Chinese hamster ovary (CHO-K1) cells for protein and cell biological analysis. Biosynthetic studies revealed that Y210C 4-sulfatase was synthesized at a comparable molecular size and amount to wild-type 4-sulfatase, but there was evidence of delayed processing, traffic, and stability of the mutant protein. Thirty-three percent of the intracellular Y210C 4-sulfatase remained as a precursor form, for at least 8 h post labeling and was not processed to the mature lysosomal form. However, unlike other 4-sulfatase mutations causing MPS VI, a significant amount of Y210C 4-sulfatase escaped the endoplasmic reticulum and was either secreted from the expression cells or underwent delayed intracellular traffic. Sixty-seven percent of the intracellular Y210C 4-sulfatase was processed to the mature form (43, 8, and 7 kDa molecular mass forms) by a proteolytic processing step known to occur in endosomes-lysosomes. Treatment of Y210C CHO-K1 cells with the protein stabilizer glycerol resulted in increased amounts of Y210C 4-sulfatase in endosomes, which was eventually trafficked to the lysosome after a long, 24 h chase time. This demonstrated delayed traffic of Y210C 4-sulfatase to the lysosomal compartment. The endosomal Y210C 4-sulfatase had a low specific activity, suggesting that the mutant protein also had problems with stability. Treatment of Y210C CHO-K1 cells with the protease inhibitor ALLM resulted in an increased amount of mature Y210C 4-sulfatase localized in lysosomes, but this protein had a very low level of activity. This indicated that the mutant protein was being inactivated and degraded at an enhanced rate in the lysosomal compartment. Biochemical analysis of Y210C 4-sulfatase revealed a normal pH optimum for the mutant protein but demonstrated a reduced enzyme activity with time, also consistent with a protein stability problem. This study indicated that multiple subcellular and biochemical processes can contribute to the biogenesis of mutant protein and may in turn influence the clinical phenotype of a patient. In MPS VI patients with a Y210C allele, the composite effect of different stages of intracellular processing/handling and environment has been shown to cause a reduced level of Y210C 4-sulfatase protein and activity, resulting in an attenuated clinical phenotype.

Mucopolysaccharidosis type VI (MPS-VI) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-4-sulfatase (4S; or ARSB). Mutations in the 4S gene are responsible for 4S deficiency, which leads to the intralysosomal storage of partially degraded glycosaminoglycans, dermatan sulfate, and chondroitin 4-sulfate. To date, a total of 45 clinically relevant mutations have been identified in the human 4S gene. Missense mutations are the largest group, with 31 identified mutations. Nonsense mutations and small insertions or deletions comprise the remainder, with seven mutations each. Six polymorphisms have also been reported: two amino acid substitutions and four silent transitions. Mapping of the missense mutations onto the 4S structure shows that they are distributed throughout the three subunits of the mature 4S polypeptide. Mutations have been identified in active site residues, in residues adjacent to the active site, in potential substrate binding residues, in residues exposed on the surface, and in residues buried within the protein core. Missense mutations have also been identified in disulfide crosslinks. Molecular modeling of MPS-VI mutations onto the 4S structure suggests that the majority cause 4S deficiency via destabilization and the consequent reduction of 4S protein concentration. The vast majority of MPS-VI mutant alleles are either unique to a patient or are present in a small number of patients. So far, no common mutations have been described. Therefore, screening of the general population for MPS-VI alleles will be difficult. Hum Mutat 18:282–295, 2001. © 2001 Wiley-Liss, Inc.