Timothy Lockwich

Trp1-dependent enhancement of salivary gland fluid secretion: role of store-operated calcium entry

This study examined the involvement of store‐operated Ca2+ entry in agonist‐stimulation of salivary gland fluid secretion. A recombinant adenovirus (AdCMV‐hTrp1) encoding the store‐operated Ca2+ channel protein, human transient receptor potential 1 (hTrp1), was used to direct expression of HA (hemaglutinin )‐tagged hTrp1 in vivo in rat submandibular glands (SMG) and in vitro in the human submandibular gland cell line (HSG). Studies with HSG cells demonstrated that AdCMV‐hTrp1 was successful in directing the expression of functional hTrp1 and that it did not affect early Ca2+ signaling events. AdCMV‐hTrp1‐infected SMG displayed an increase in the level of Trp1 and a fivefold increase in pilocarpine‐stimulated fluid secretion, compared with glands infected with a control adenovirus encoding luciferase (AdCMV‐Luc). The expressed hTrp1 demonstrated polarized localization in the basolateral plasma membrane region of SMG acinar cells and was co‐immunoprecipitated with IP3Rs. Further, acinar cells isolated from AdCMV‐hTrp1‐infected glands demonstrated a significant increase in carbachol‐ and Tg‐stimulated Ca2+ entry compared with cells isolated from AdCMV‐Luc‐infected glands. We conclude that in vivo expression of Trp1 in SMG induces an enhancement of agonist‐stimulated fluid secretion via increasing store‐operated Ca2+ entry into acinar cells. These data suggest that store‐operated Ca2+ entry has a role in agonist‐stimulated fluid secretion from salivary glands.

TRPC3 Controls Agonist-stimulated Intracellular Ca2+ Release by Mediating the Interaction between Inositol 1,4,5-Trisphosphate Receptor and RACK1

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated intracellular Ca2+ release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP3R with RACK1 and IP3-dependent intracellular Ca2+ release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP3R complex and increased surface expression of TRPC3 and Ca2+ entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP3R complex as well as increased surface expression of TRPC3 and receptor-operated Ca2+ entry were also attenuated. Importantly, CCh-induced intracellular Ca2+ release was significantly reduced as was RACK1-IP3R association without any change in thapsigargin-stimulated Ca2+ release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP3R association and decreased CCh-stimulated Ca2+ entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca2+ release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca2+ release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP3R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating interaction between IP3R and RACK1.

Analysis of TRPC3-interacting proteins by tandem mass spectrometry.

Mammalian transient receptor potential canonical (TRPC) channels are a family of nonspecific cation channels that are activated in response to stimulation of phospholipase C (PLC)-dependent hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. Despite extensive studies, the mechanism(s) involved in regulation of mammalian TRPC channels remains unknown. Presence of various protein-interacting domains in TRPC channels have led to the suggestion that they associate with proteins that are involved in their function and regulation. This study was directed toward identifying the proteins associated with native TRPC3 using a shotgun proteomic approach. Anti-TRPC3 antibody was used to immunoprecipitate TRPC3 from solubilized rat brain crude membranes under conditions that allow retention of TRPC3 function. Proteins in the TRPC3 (using anti-TRPC3 antibody) and control (using rabbit IgG) immunoprecipitates were separated by SDS-PAGE, the gel was sectioned, and the resolved proteins were digested by trypsin in situ. After extraction of the peptides, the peptides were separated by HPLC and sequences derived by MS/MS. Analysis of the data revealed 64 specific TRPC3-associated proteins which can be grouped in terms of their cellular location and involvement in specific cellular function. Many of the proteins identified have been previously reported as TRPC3-regulatory proteins, such as IP3Rs and vesicle trafficking proteins. In addition, we report novel putative TRPC3-interacting proteins, including those involved in protein endocytosis and neuronal growth. To our knowledge, this is the first comprehensive proteomic analysis of a native TRPC channel. These data reveal potential TRPC3 regulatory proteins and provide novel insights of the mechanism(s) regulating TRPC3 channels as well as the possible cellular functions where the channel might be involved.

Calcium entry in rat parotid acinar cells

ConclusionsWhile it is generally accepted that Ca2+ plays an important regulatory role in the physiology of a number of non-excitable cells, the mechanisms which regulate intracellular [Ca2+ are far from well established. Ca2+ transporting mechanisms which distribute Ca2+ intracellularly as well as those which allow influx of extracellular Ca2+ are involved in mediating intracellular Ca2+ homestasis. In this paper we have described recent studies on the regulation of the Ca2+ influx system in the data, it appears that the process of Ca2+ entry is extremely complex and may involve several levels of regulation. Understanding the molecular basis of these regulatory mechanisms presents a challeging problem for future studies.

Reconstitution of a passive Ca2+-transport pathway from the basolateral plasma membrane of rat parotid gland acinar cells

We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca2+ transport pathway and an unsaturable Ca2+ flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289–296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3–5-fold higher 45Ca2+ influx than control liposomes (without protein). Ca2+ uptake into PrL was dependent on the [protein] in PrL and steady state [Ca2+] in PrL was in equilibrium with external [Ca2+]. These data demonstrate that a passive, protein-mediated Ca2+ transport has been reconstituted from BLMV into PrL. 45Ca2+ influx into liposomes did not saturate with increasing [Ca2+] in the assay medium. In contrast, PrL displayed saturable 45Ca2+ influx and exhibited a single Ca2+ flux component with an apparent Kca=242 ± 50.9 μm and Vmax=13.5 ± 1.14 nmoles Ca2+/mg protein/ minute. The Kca of Ca2+-transport in PrL was similar to that of the high affinity Ca2+ influx component in BLMV while the Vmax was about 4-fold higher. The unsaturable Ca2+ flux component was not detected in PrL. 45Ca2+ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn2+>Mn2+>Co2+=Ni2+, and appeared to be more sensitive to lower concentrations of Zn2+ than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N′-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca2+ transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40–50% decrease in Vmax of Ca2+ transport without an alteration in Kca. These data strongly suggest that the high affinity, passive Ca2+ transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca2+ influx in the rat parotid gland basolateral plasma membrane.

Involvement of carboxyl groups in the divalent cation permeability of rat parotid gland basolateral plasma membrane

Divalent cation permeability of rat parotid gland basolateral plasma membranes was examined in dispersed parotid acini (by Ca2+ or Mn2+ entry) and in isolated basolateral plasma membrane vesicles (BLMV, by45Ca2+ influx). Mn2+ entry (fura2 quenching) was about 1.6 fold higher in internal Ca2+ pool-depleted acini (Ca2+-depl acini) than in unstimulated cells. Mn2+ entry into Ca2+-depl acini was increased at external pH>7.4 and decreased at pH<7.4. Pretreatment of Ca2+-depl acini with the relatively hydrophobic carboxylic group reagent, N,N′-dicyclohexylcarbodiimide (DCCD, 50 μM for 30 min) resulted in the inhibition of Mn2+ entry into Ca2+-depl acini to unstimulated levels. Another hydrophobic carboxyl group reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and the relatively hydrophilic carboxyl group reagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMCD) did not affect Mn2+ entry.Similar to the effects in intact acini, Ca2+ influx into BLMV was decreased when the external pH was lowered below 7.4. Also DCCD (5 mM, 30 min), but not EEDQ, decreased (40%) Ca2+ influx in BLMV. However, unlike in acini, the hydrophilic reagents, EDC, EAC, and CMCD decreased Ca2+ permeability in BLMV and the effects were nonadditive with the decrease induced by DCCD. The aggregate effects of carboxyl group reagents on the Ca2+ and Mn2+ permeability in BLMV and intact acini, respectively, suggest that a critical carboxyl group (most likely accessible from the cytoplasmic side of the plasma membrane) is involved in divalent cation flux in rat parotid acinar cells.

CHARACTERISTICS OF A LOW AFFINITY PASSIVE CA2+ INFLUX COMPONENT IN RAT PAROTID GLAND BASOLATERAL PLASMA MEMBRANE VESICLES

Abstract. We have previously reported the presence of two Ca2+ influx components with relatively high (KCa= 152 ± 79 μm) and low (KCa= 2.4 ± 0.9 mm) affinities for Ca2+ in internal Ca2+ pool-depleted rat parotid acinar cells [Chauthaiwale et al. (1996) Pfluegers Arch. 432:105–111]. We have also reported the presence of a high affinity Ca2+ influx component with KCa= 279 ± 43 μm in rat parotid gland basolateral plasma membrane vesicles (BLMV). [Lockwich, Kim & Ambudkar (1994) J. Membrane Biol. 141:289–296]. The present studies show that a low affinity Ca2+ influx component is also present in BLMV with KCa= 2.3 ± 0.41 mm (Vmax= 16.36 ± 4.11 nmoles of Ca2+/mg protein/min). Our data demonstrate that this low affinity component is similar to the low affinity Ca2+ influx component that is activated by internal Ca2+ store depletion in dispersed parotid gland acini by the following criteria: (i) similar KCa for calcium flux, (ii) similar IC50 for inhibition by Ni2+ and Zn2+; (iii) increase in KCa at high external K+, (iv) similar effects of external pH. The high affinity Ca2+ influx in cells is different from the low affinity Ca2+ influx component cells in its sensitivity to pH, KCl, Zn2+ and Ni2+. The low and high affinity Ca2+ influx components in BLMV can also be distinguished from each other based on the effects of Zn2+, Ni2+, KCl, and dicyclohexylcarbodiimide. In aggregate, these data demonstrate the presence of a low affinity passive Ca2+ influx pathway in BLMV which displays characteristics similar to the low affinity Ca2+ influx component detected in parotid acinar cells following internal Ca2+ store depletion.