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When the K + channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

Background and Purpose: The rise in intracellular Ca(2+) stimulates the expression of the transcription factor c-Fos. Depending on the mode of entry of Ca(2+) into the cytosol, distinct signal transducers and transcription factors are required. Here, we have analysed the signalling pathway connecting a Ca(2+) influx via activation of transient receptor potential melastatin-3 (TRPM3) channels with enhanced c-Fos expression., Experimental Approach: Transcription of c-Fos promoter/reporter genes that were integrated into the chromatin via lentiviral gene transfer was analysed in HEK293 cells overexpressing TRPM3. The transcriptional activation potential of c-Fos was measured using a GAL4-c-Fos fusion protein., Key Results: The signalling pathway connecting TRPM3 stimulation with enhanced c-Fos expression requires the activation of MAP kinases. On the transcriptional level, three Ca(2+) -responsive elements, the cAMP-response element and the binding sites for the serum response factor (SRF) and AP-1, are essential for the TRPM3-mediated stimulation of the c-Fos promoter. Ternary complex factors are additionally involved in connecting TRPM3 stimulation with the up-regulation of c-Fos expression. Stimulation of TRPM3 channels also increases the transcriptional activation potential of c-Fos., Conclusions and Implications: Signalling molecules involved in connecting TRPM3 with the c-Fos gene are MAP kinases and the transcription factors CREB, SRF, AP-1 and ternary complex factors. As c-Fos constitutes, together with other basic region leucine zipper transcription factors, the AP-1 transcription factor complex, the results of this study explain TRPM3-induced activation of AP-1 and connects TRPM3 with the biological functions regulated by AP-1., (© 2015 The British Pharmacological Society.)

Fusion of exocytotic vesicles with the plasma membrane gives rise to an increase in membrane surface area, whereas the surface area is decreased when vesicles are internalized during endocytosis. Changes in membrane surface area, resulting from fusion and fission of membrane vesicles, can be followed by monitoring the corresponding proportional changes in membrane capacitance. Using the cell-attached configuration of the patch-clamp techniques we were able to resolve the elementary processes of endo- and exocytosis in yeast protoplasts at high temporal and spatial resolution. Spontaneous capacitance changes were predominantly in the range of 0.2-1 fF which translates to vesicle diameters of 90-200 nm. The size distribution revealed that endocytotic vesicles with a median at about 132 nm were smaller than exocytotic vesicles with a median at 155 nm. In energized and metabolizing protoplasts, endo- and exocytotic events occurred at frequencies of 1.6 and 2.7 events per minute, respectively. Even though these numbers appear very low, they are in good agreement with the observed growth rate of yeast cells and protoplasts., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Purpose: Transient receptor potential-3 (TRPM3) channels function as Ca2+ permeable cation channels. While the natural ligands for these channels are still unknown, several compounds have been described that either activate or inhibit TRPM3 channel activity. experimental approach: We assessed TRPM3-mediated gene transcription, which relies on the induction of intracellular signalling to the nucleus following activation of TRPM3 channels. Activator protein-1 (AP-1) and Egr-1-responsive reporter genes were integrated into the chromatin of the cells. This strategy enabled us to analyse gene transcription of the AP-1 and Egr-1-responsive reporter genes that were packed into an ordered chromatin structure., Key Results: The neurosteroid pregnenolone sulfate strikingly up-regulated AP-1 and Egr-1 transcriptional activity, while nifedipine and D-erythro-sphingosine, also putative activators of TRPM3 channels, exhibited either no or TRPM3-independent effects on gene transcription. In addition, pregnenolone sulfate robustly enhanced the transcriptional activation potential of the ternary complex factor Elk-1. Pregnenolone sulfate-induced activation of gene transcription was blocked by treatment with mefenamic acid and, to a lesser extent, by the polyphenol naringenin. In contrast, progesterone, pregnenolone and rosiglitazone reduced AP-1 activity in the cells, but had no inhibitory effect on Egr-1 activity in pregnenolone sulfate-stimulated cells., Conclusion and Implications: Pregnenolone sulfate is a powerful activator of TRPM3-mediated gene transcription, while transcription is completely inhibited by mefenamic acid in cells expressing activated TRPM3 channels. Both compounds are valuable tools for further investigating the biological functions of TRPM3 channels.

Using the bright, photostable, charged and hydrophilic fluorescent dye Alexa 488 hydrazide to label the fluid phase around intact guard cells, we show that these cells incorporate the fluid phase during constitutive endocytosis against the high turgor. Mobile, cortical and diffraction-limited signals were not observed if a concentration <4 mm was used to stain the fluid phase, suggesting that endocytic vesicles had to be loaded with a minimal number of dye molecules to produce a signal above the background. To quantify the number of molecules taken up by the vesicles, we prepared liposomes, filled with various concentrations of Alexa 488 hydrazide, fractionated them according to their size and imaged them under identical conditions as the guard cells. From the size/intensity relations of these liposomes, we extrapolated the molecular brightness of Alexa 488 hydrazide. Using this calibration, the mean fluorescent intensity of single endocytic vesicles translates into a mean number of 573 Alexa 488 molecules. If a vesicle needs to take up 573 molecules from a 4 mm solution, it requires a diameter of at least 87 nm. This number provides the first in vivo estimate for the size of endocytic vesicles in intact, turgid plant cells.

PPI1 (proton pump interactor isoform 1) is a novel protein able to interact with the C-terminal autoinhibitory domain of the Arabidopsis thaliana plasma membrane (PM) H(+)-ATPase. In vitro, PPI1 binds the PM H(+)-ATPase in a site different from the known 14-3-3 binding site and stimulates its activity. In this study, we analysed the intracellular localisation of PPI1. The intracellular distribution was monitored in A. thaliana cultured cells by immunolocalisation using an antiserum against the PPI1 N-terminus and in Vicia faba guard cells and epidermal cells by transient expression of a GFP::PPI1 fusion. The results indicate that the bulk of PPI1 is localised at the endoplasmic reticulum, from which it might be recruited to the PM for interaction with the H(+)-ATPase in response to as yet unidentified signals.

Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC(50) values: 0.2-8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug-drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6beta-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S-mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O-dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 microM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC(50) values around 200 microM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC(50) value: 4 microM). Using the assay of testosterone 6beta-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC(50) values of about 200 microM and a little more by C and A (IC(50) around 100 microM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug-drug interaction is considered., (Copyright 2000 John Wiley & Sons, Ltd.)

Kinetics of the novel immunosuppressive cyclosporine were determined in four patients with terminal renal failure. After a short intravenous infusion (2.05 to 3.5 mg/kg in 4 hr), blood and plasma concentrations were measured (HPLC and radioimmunoassay [RIA] up to 36 hr. After infusion, concentration curves of the drug were characterized by a rapid initial fall (t 1/2 alpha 0.10 +/- 0.03 hr), followed by a biphasic elimination phase with corresponding t 1/2s of 1.08 +/- 0.25 hr (t 1/2 beta) and 15.8 +/- 8.4 hr (t 1/2 gamma). The volumes of distribution, calculated from whole blood concentrations (HPLC), were 0.140 +/- 0.48 l/kg (volume of the central compartment) and 3.49 +/- 2.65 l/kg (volume of distribution at steady state), whereas systemic clearances were 0.369 +/- 0.08 l/hr/kg. Blood levels measured by RIA exceeded the HPLC values after the fourth hour by up to 100%, indicating the production of cross-reacting cyclosporine metabolites. Plasma concentrations were considerably lower than in whole blood. Elimination of unchanged cyclosporine in patients with renal failure appears to be of the same order as in those with normal kidney function. Modification of the initial dosage regimens is therefore probably not required.