Different classes of ion channels have been implicated in sensing cold temperatures at mammalian ... more Different classes of ion channels have been implicated in sensing cold temperatures at mammalian thermoreceptor nerve endings. A major candidate is TRPM8, a non-selective cation channel of the transient receptor potential family, activated by menthol and low temperatures. We investigated the role of TRPM8 in cold sensing during transient expression in mouse cultured hippocampal neurones, a tissue that lacks endogenous expression of thermosensitive TRPs. In the absence of synaptic input, control hippocampal neurones were not excited by cooling. In contrast, all TRPM8-transfected hippocampal neurones were excited by cooling and menthol. However, in comparison to cold-sensitive trigeminal sensory neurones, hippocampal neurones exhibited much lower threshold temperatures, requiring temperatures below 27 • C to fire action potentials. These results directly demonstrate that expression of TRPM8 in mammalian neurones induces cold sensing, albeit at lower temperatures than native TRPM8-expressing neurones, suggesting the presence of additional modulatory mechanisms in the cold response of sensory neurones.
Background: TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of t... more Background: TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of the transient receptor potential (TRP) ion channels. TRPM8 is activated by voltage, cold and cooling compounds such as menthol. Despite its essential role for cold temperature sensing in mammals, the pharmacology of TRPM8 is still in its infancy. Recently, tyrosine 745 (Y745) was identified as a critical residue for menthol sensitivity of the channel. In this report, we study the effect of mutating this residue on the action of several known TRPM8 antagonists: BCTC, capsazepine, SKF96365, and clotrimazole as well as two new inhibitor candidates, econazole and imidazole.
TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels... more TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels, is activated by voltage, low temperatures and cooling compounds. These properties and its restricted expression to small sensory neurons have made it the ion channel with the most advocated role in cold transduction. Recent work suggests that activation of TRPM8 by cold and menthol takes place through shifts in its voltage-activation curve, which cause the channel to open at physiological membrane potentials. By contrast, little is known about the actions of inhibitors on the function of TRPM8. We investigated the chemical and thermal modulation of TRPM8 in transfected HEK293 cells and in cold-sensitive primary sensory neurons. We show that cold-evoked TRPM8 responses are effectively suppressed by inhibitor compounds SKF96365, 4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide (BCTC) and 1,10-phenanthroline. These antagonists exert their effect by shifting the voltage dependence of TRPM8 activation towards more positive potentials. An opposite shift towards more negative potentials is achieved by the agonist menthol. Functionally, the bidirectional shift in channel gating translates into a change in the apparent temperature threshold of TRPM8-expressing cells. Accordingly, in the presence of the antagonist compounds, the apparent response-threshold temperature of TRPM8 is displaced towards colder temperatures, whereas menthol sensitizes the response, shifting the threshold in the opposite direction. Co-application of agonists and antagonists produces predictable cancellation of these effects, suggesting the convergence on a common molecular process. The potential for half maximal activation of TRPM8 activation by cold was ∼140 mV more negative in native channels compared to recombinant channels, with a much higher open probability at negative membrane potentials in the former. In functional terms, this difference translates into a shift in the apparent temperature threshold for activation towards higher temperatures for native currents. This difference in voltage-dependence readily explains the high threshold temperatures characteristic of many cold thermoreceptors. The modulation of TRPM8 activity by different chemical agents unveils an important flexibility in the temperature-response curve of TRPM8 channels and cold thermoreceptors.
Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by ... more Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by cold temperature and cooling agents. TRPM8 is expressed in a subpopulation of cold-sensitive sensory neurons, as well as in the male urogenital system. TRPM8 is markedly upregulated in prostate cancer and in other tumors such as breast adenocarcinoma and melanoma. Moreover, recent studies suggest the potential involvement of TRPM8 channels in the pathophysiology of cold nociception and cold allodynia. This has led to a strong interest in the pursuit of novel modulators of TRPM8 channels. This review highlights our current knowledge of TRPM8 pharmacology and modulation mechanisms, detailing structural features important for TRPM8 gating by different agonists, the mechanism of antagonism by different compounds and the potential relevance of TRPM8 for treatment of various pathological conditions.
Drug Transfer Through Biomimetic Langmuir-Blodgett Monolayers at a Liquid-Liquid Interface
Analytical Sciences
The effect of cholesterol and lipid composition on the properties of phospholipid monolayers at a... more The effect of cholesterol and lipid composition on the properties of phospholipid monolayers at a liquid-liquid interface have been investigated with impedance spectroscopy. Drug transfer through lipid layers of varying composition has been interpreted with the aid of a model where an adsorption step has been coupled to charge transfer. This yields information on the interactions of the drug with both the aqueous and the organic sides of the monolayer. The results indicate that changing the composition of the layer has a relatively minor effect on the electrochemical response of the system. The most significant effect of adding cholesterol is to decrease the adsorption of the lipophilic drug on the aqueous side of the monolayer.
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 2004
In the past decades, it has become increasingly apparent that in addition to therapeutic effect, ... more In the past decades, it has become increasingly apparent that in addition to therapeutic effect, drugs need to exhibit favourable absorption, distribution, metabolism and excretion (ADME) characteristics to produce a desirable response in vivo. As the recent progress in drug discovery technology enables rapid synthesis of vast numbers of potential drug candidates, robust methods are required for the effective screening of compounds synthesized within such programs, so that compounds with poor pharmacokinetic properties can be rejected at an early stage of drug development. Furthermore, a viable in silico method would save resources by enabling virtual screening of drug candidates already prior to synthesis. This review gives a general overview of the approaches aimed at predicting biological permeation, one of the cornerstones behind the ADME behaviour of drugs. The most important experimental and computational models are reviewed. Physicochemical factors underlying the permeation p...
Different classes of ion channels have been implicated in sensing cold temperatures at mammalian ... more Different classes of ion channels have been implicated in sensing cold temperatures at mammalian thermoreceptor nerve endings. A major candidate is TRPM8, a non-selective cation channel of the transient receptor potential family, activated by menthol and low temperatures. We investigated the role of TRPM8 in cold sensing during transient expression in mouse cultured hippocampal neurones, a tissue that lacks endogenous expression of thermosensitive TRPs. In the absence of synaptic input, control hippocampal neurones were not excited by cooling. In contrast, all TRPM8-transfected hippocampal neurones were excited by cooling and menthol. However, in comparison to cold-sensitive trigeminal sensory neurones, hippocampal neurones exhibited much lower threshold temperatures, requiring temperatures below 27 • C to fire action potentials. These results directly demonstrate that expression of TRPM8 in mammalian neurones induces cold sensing, albeit at lower temperatures than native TRPM8-expressing neurones, suggesting the presence of additional modulatory mechanisms in the cold response of sensory neurones.
TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels... more TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels, is activated by voltage, low temperatures and cooling compounds. These properties and its restricted expression to small sensory neurons have made it the ion channel with the most advocated role in cold transduction. Recent work suggests that activation of TRPM8 by cold and menthol takes place through shifts in its voltage-activation curve, which cause the channel to open at physiological membrane potentials. By contrast, little is known about the actions of inhibitors on the function of TRPM8. We investigated the chemical and thermal modulation of TRPM8 in transfected HEK293 cells and in cold-sensitive primary sensory neurons. We show that cold-evoked TRPM8 responses are effectively suppressed by inhibitor compounds SKF96365, 4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide (BCTC) and 1,10-phenanthroline. These antagonists exert their effect by shifting the voltage dependence of TRPM8 activation towards more positive potentials. An opposite shift towards more negative potentials is achieved by the agonist menthol. Functionally, the bidirectional shift in channel gating translates into a change in the apparent temperature threshold of TRPM8-expressing cells. Accordingly, in the presence of the antagonist compounds, the apparent response-threshold temperature of TRPM8 is displaced towards colder temperatures, whereas menthol sensitizes the response, shifting the threshold in the opposite direction. Co-application of agonists and antagonists produces predictable cancellation of these effects, suggesting the convergence on a common molecular process. The potential for half maximal activation of TRPM8 activation by cold was ∼140 mV more negative in native channels compared to recombinant channels, with a much higher open probability at negative membrane potentials in the former. In functional terms, this difference translates into a shift in the apparent temperature threshold for activation towards higher temperatures for native currents. This difference in voltage-dependence readily explains the high threshold temperatures characteristic of many cold thermoreceptors. The modulation of TRPM8 activity by different chemical agents unveils an important flexibility in the temperature-response curve of TRPM8 channels and cold thermoreceptors.
Electrochemistry at Lipid Monolayer-Modified Liquid−Liquid Interfaces as an Improvement to Drug Partitioning Studies
The Journal of Physical Chemistry B, 2001
... Annika Mälkiä, Peter Liljeroth, Anna-Kaisa Kontturi, and Kyösti Kontturi*. Laboratory of Phys... more ... Annika Mälkiä, Peter Liljeroth, Anna-Kaisa Kontturi, and Kyösti Kontturi*. Laboratory of Physical Chemistry and Electrochemistry, Helsinki University of Technology, PO Box 6100, FIN-02015 HUT, Finland. J. Phys. Chem. B , 2001 ...
The hippocampus plays an important role in short term memory, learning and spatial navigation. A ... more The hippocampus plays an important role in short term memory, learning and spatial navigation. A characteristic feature of the hippocampal region is its expression of different electrical population rhythms and activities during different brain states. Physiological fluctuations in brain temperature affect the activity patterns in hippocampus, but the underlying cellular mechanisms are poorly understood. In this work, we investigated the thermal modulation of hippocampal activity at the cellular network level. Primary cell cultures of mouse E17 hippocampus displayed robust network activation upon light cooling of the extracellular solution from baseline physiological temperatures. The activity generated was dependent on action potential firing and excitatory glutamatergic synaptic transmission. Involvement of thermosensitive channels from the transient receptor potential (TRP) family in network activation by temperature changes was ruled out, whereas pharmacological and immunochemical experiments strongly pointed towards the involvement of temperature-sensitive two-pore-domain potassium channels (K 2P ), TREK/TRAAK family. In hippocampal slices we could show an increase in evoked and spontaneous synaptic activity produced by mild cooling in the physiological range that was prevented by chloroform, a K 2P channel opener. We propose that cold-induced closure of background TREK/TRAAK family channels increases the excitability of some hippocampal neurons, acting as a temperature-sensitive gate of network activation. Our findings in the hippocampus open the possibility that small temperature variations in the brain in vivo, associated with metabolism or blood flow oscillations, act as a switch mechanism of neuronal activity and determination of firing patterns through regulation of thermosensitive background potassium channel activity.
Treatment with resveratrol (RSV) has been shown to protect vulnerable neurons after various brain... more Treatment with resveratrol (RSV) has been shown to protect vulnerable neurons after various brain injuries and in neurodegenerative diseases. The mechanisms for the effects of RSV in brain are not fully understood, but RSV may affect the expression of various gene products. RSV is structurally related to the synthetic estrogen, diethylstilbestrol so the effects of RSV may be gender-specific. Here we studied the role of RSV in the regulation of dopamine transporter (DAT) in the striatum using male and female mice. The basic levels of DAT in the striatum showed no sex difference, but the levels increased significantly by RSV (20 mg/kg i.p.) in female but not in male mice. Pretreatment of mice with the selective estrogen receptor (ER), ERa-and ERb antagonist ICI 182,780, led to a complete block of RSV effect on DAT protein levels, suggesting that ERs are involved in the up-regulation of DAT by RSV. Similar data was also obtained in culture using human MESC2.10 and mouse SN4741 dopaminergic cells after treatment with RSV. Data further showed that RSV specifically induced gene transcription of DAT in the dopaminergic cells. These results show that estrogen receptors are involved in the up-regulation of DAT by RSV in the dopaminergic neurons, demonstrating a sex-dependent effect of RSV in the brain that may be of clinical importance.
Background: TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of t... more Background: TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of the transient receptor potential (TRP) ion channels. TRPM8 is activated by voltage, cold and cooling compounds such as menthol. Despite its essential role for cold temperature sensing in mammals, the pharmacology of TRPM8 is still in its infancy. Recently, tyrosine 745 (Y745) was identified as a critical residue for menthol sensitivity of the channel. In this report, we study the effect of mutating this residue on the action of several known TRPM8 antagonists: BCTC, capsazepine, SKF96365, and clotrimazole as well as two new inhibitor candidates, econazole and imidazole.
Polyelectrolyte/gold nanoparticle multilayers composed of poly(L-lysine) (pLys) and mercaptosucci... more Polyelectrolyte/gold nanoparticle multilayers composed of poly(L-lysine) (pLys) and mercaptosuccinic acid (MSA) stabilized gold nanoparticles (Au NPs) were built up using the electrostatic layer-by-layer self-assembly technique upon a gold electrode modified with a first layer of MSA. The assemblies were characterized using UV-vis absorption spectroscopy, cyclic and square-wave voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. Charge transport through the multilayer was studied experimentally as well as theoretically by using two different redox pairs [Fe(CN) 6 ] 3-/4and [Ru(NH 3 ) 6 ] 3+/2+ . This paper reports a large sensitivity to the charge of the outermost layer for the permeability of these assemblies to the probe ions. With the former redox pair, dramatic changes in the impedance response were obtained for thin multilayers each time a new layer was deposited. In the latter case, the multilayer behaves as a conductor exhibiting a strikingly lower impedance response, the electric current being enhanced as more layers are added for Au NP terminated multilayers. These results are interpreted quite satisfactorily by means of a capillary membrane model that encompasses the wide variety of behaviors observed. It is concluded that nonlinear slow diffusion through defects (pinholes) in the multilayer is the governing mechanism for the [Fe(CN) 6 ] 3-/4species, whereas electron transfer through the Au NPs is the dominant mechanism in the case of the [Ru(NH 3 ) 6 ] 3+/2+ pair.
Structurally related aminoacridine therapeutics (aminacrine, tacrine, velnacrine and proflavine) ... more Structurally related aminoacridine therapeutics (aminacrine, tacrine, velnacrine and proflavine) were studied electrochemically at the interface between two immiscible electrolyte solutions. To assess both their neutral and ionic partition coefficients, cyclic voltammetry was used to obtain the transfer potential as a function of the aqueous phase pH. Subsequently, in order to create a simple model of a biological membrane, a lipid monolayer was deposited at the liquid-liquid interface using the Langmuir-Blodgett technique. Electrochemical impedance spectroscopy was employed to study the interactions between the drug ions and the biomimetic phospholipid monolayer. Using a model incorporating a charge transfer mechanism coupled with potential dependent adsorption steps, the membrane activity of the studied drug ions could be estimated. The results indicate liquid-liquid electrochemistry to be a versatile tool for detecting differences in membrane activity even between structurally very similar drugs.
Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by ... more Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by cold temperature and cooling agents. TRPM8 is expressed in a subpopulation of cold-sensitive sensory neurons, as well as in the male urogenital system. TRPM8 is markedly upregulated in prostate cancer and in other tumors such as breast adenocarcinoma and melanoma. Moreover, recent studies suggest the potential involvement of TRPM8 channels in the pathophysiology of cold nociception and cold allodynia. This has led to a strong interest in the pursuit of novel modulators of TRPM8 channels. This review highlights our current knowledge of TRPM8 pharmacology and modulation mechanisms, detailing structural features important for TRPM8 gating by different agonists, the mechanism of antagonism by different compounds and the potential relevance of TRPM8 for treatment of various pathological conditions.
This thesis describes the development and characterisation of a novel biomembrane model suitable ... more This thesis describes the development and characterisation of a novel biomembrane model suitable for electrochemical investigation, and its applicability in studies of biologically related phenomena. The model system consists of a lipid monolayer, deposited at a polarisable liquid -liquid interface with the Langmuir -Blodgett technique. Combinations of ac and dc electrochemical techniques with theoretical models are employed to obtain information on the phase transfer and membrane interactions of charged therapeutics at the monolayer-covered interface. The thesis comprises six publications and a thorough introduction to biological and model membranes.
Membrane activity of biotechnological peptide drugs
Chemical Communications, 2003
Charged Langmuir-Blodgett monolayers deposited at an immobilised liquid-liquid interface have bee... more Charged Langmuir-Blodgett monolayers deposited at an immobilised liquid-liquid interface have been used as a simple model for a biological membrane to investigate the membrane activity of biotechnological oligopeptide drugs.
Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but little is k... more Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but little is known about the molecular mechanisms controlling these events. Peroxisome proliferator-activated receptor-gamma coactivator-1a (PGC-1a) is a transcriptional coactivator that is a master regulator of oxidative stress and mitochondrial metabolism. We show here that transgenic mice overexpressing PGC-1a in dopaminergic neurons are resistant against cell degeneration induced by the neurotoxin MPTP. The increase in neuronal viability was accompanied by elevated levels of mitochondrial antioxidants SOD2 and Trx2 in the substantia nigra of transgenic mice. PGC-1a overexpression also protected against MPTP-induced striatal loss of dopamine, and mitochondria from PGC-1a transgenic mice showed an increased respiratory control ratio compared with wild-type animals. To modulate PGC-1a, we employed the small molecular compound, resveratrol (RSV) that protected dopaminergic neurons against the MPTP-induced cell degeneration almost to the same extent as after PGC-1a overexpression. As studied in vitro, RSV activated PGC-1a in dopaminergic SN4741 cells via the deacetylase SIRT1, and enhanced PGC-1a gene transcription with increases in SOD2 and Trx2. Taken together, the results reveal an important function of PGC-1a in dopaminergic neurons to combat oxidative stress and increase neuronal viability. RSV and other compounds acting via SIRT1/PGC-1a may prove useful as neuroprotective agents in PD and possibly in other neurological disorders.
Different classes of ion channels have been implicated in sensing cold temperatures at mammalian ... more Different classes of ion channels have been implicated in sensing cold temperatures at mammalian thermoreceptor nerve endings. A major candidate is TRPM8, a non-selective cation channel of the transient receptor potential family, activated by menthol and low temperatures. We investigated the role of TRPM8 in cold sensing during transient expression in mouse cultured hippocampal neurones, a tissue that lacks endogenous expression of thermosensitive TRPs. In the absence of synaptic input, control hippocampal neurones were not excited by cooling. In contrast, all TRPM8-transfected hippocampal neurones were excited by cooling and menthol. However, in comparison to cold-sensitive trigeminal sensory neurones, hippocampal neurones exhibited much lower threshold temperatures, requiring temperatures below 27 • C to fire action potentials. These results directly demonstrate that expression of TRPM8 in mammalian neurones induces cold sensing, albeit at lower temperatures than native TRPM8-expressing neurones, suggesting the presence of additional modulatory mechanisms in the cold response of sensory neurones.
Background: TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of t... more Background: TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of the transient receptor potential (TRP) ion channels. TRPM8 is activated by voltage, cold and cooling compounds such as menthol. Despite its essential role for cold temperature sensing in mammals, the pharmacology of TRPM8 is still in its infancy. Recently, tyrosine 745 (Y745) was identified as a critical residue for menthol sensitivity of the channel. In this report, we study the effect of mutating this residue on the action of several known TRPM8 antagonists: BCTC, capsazepine, SKF96365, and clotrimazole as well as two new inhibitor candidates, econazole and imidazole.
TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels... more TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels, is activated by voltage, low temperatures and cooling compounds. These properties and its restricted expression to small sensory neurons have made it the ion channel with the most advocated role in cold transduction. Recent work suggests that activation of TRPM8 by cold and menthol takes place through shifts in its voltage-activation curve, which cause the channel to open at physiological membrane potentials. By contrast, little is known about the actions of inhibitors on the function of TRPM8. We investigated the chemical and thermal modulation of TRPM8 in transfected HEK293 cells and in cold-sensitive primary sensory neurons. We show that cold-evoked TRPM8 responses are effectively suppressed by inhibitor compounds SKF96365, 4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide (BCTC) and 1,10-phenanthroline. These antagonists exert their effect by shifting the voltage dependence of TRPM8 activation towards more positive potentials. An opposite shift towards more negative potentials is achieved by the agonist menthol. Functionally, the bidirectional shift in channel gating translates into a change in the apparent temperature threshold of TRPM8-expressing cells. Accordingly, in the presence of the antagonist compounds, the apparent response-threshold temperature of TRPM8 is displaced towards colder temperatures, whereas menthol sensitizes the response, shifting the threshold in the opposite direction. Co-application of agonists and antagonists produces predictable cancellation of these effects, suggesting the convergence on a common molecular process. The potential for half maximal activation of TRPM8 activation by cold was ∼140 mV more negative in native channels compared to recombinant channels, with a much higher open probability at negative membrane potentials in the former. In functional terms, this difference translates into a shift in the apparent temperature threshold for activation towards higher temperatures for native currents. This difference in voltage-dependence readily explains the high threshold temperatures characteristic of many cold thermoreceptors. The modulation of TRPM8 activity by different chemical agents unveils an important flexibility in the temperature-response curve of TRPM8 channels and cold thermoreceptors.
Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by ... more Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by cold temperature and cooling agents. TRPM8 is expressed in a subpopulation of cold-sensitive sensory neurons, as well as in the male urogenital system. TRPM8 is markedly upregulated in prostate cancer and in other tumors such as breast adenocarcinoma and melanoma. Moreover, recent studies suggest the potential involvement of TRPM8 channels in the pathophysiology of cold nociception and cold allodynia. This has led to a strong interest in the pursuit of novel modulators of TRPM8 channels. This review highlights our current knowledge of TRPM8 pharmacology and modulation mechanisms, detailing structural features important for TRPM8 gating by different agonists, the mechanism of antagonism by different compounds and the potential relevance of TRPM8 for treatment of various pathological conditions.
Drug Transfer Through Biomimetic Langmuir-Blodgett Monolayers at a Liquid-Liquid Interface
Analytical Sciences
The effect of cholesterol and lipid composition on the properties of phospholipid monolayers at a... more The effect of cholesterol and lipid composition on the properties of phospholipid monolayers at a liquid-liquid interface have been investigated with impedance spectroscopy. Drug transfer through lipid layers of varying composition has been interpreted with the aid of a model where an adsorption step has been coupled to charge transfer. This yields information on the interactions of the drug with both the aqueous and the organic sides of the monolayer. The results indicate that changing the composition of the layer has a relatively minor effect on the electrochemical response of the system. The most significant effect of adding cholesterol is to decrease the adsorption of the lipophilic drug on the aqueous side of the monolayer.
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 2004
In the past decades, it has become increasingly apparent that in addition to therapeutic effect, ... more In the past decades, it has become increasingly apparent that in addition to therapeutic effect, drugs need to exhibit favourable absorption, distribution, metabolism and excretion (ADME) characteristics to produce a desirable response in vivo. As the recent progress in drug discovery technology enables rapid synthesis of vast numbers of potential drug candidates, robust methods are required for the effective screening of compounds synthesized within such programs, so that compounds with poor pharmacokinetic properties can be rejected at an early stage of drug development. Furthermore, a viable in silico method would save resources by enabling virtual screening of drug candidates already prior to synthesis. This review gives a general overview of the approaches aimed at predicting biological permeation, one of the cornerstones behind the ADME behaviour of drugs. The most important experimental and computational models are reviewed. Physicochemical factors underlying the permeation p...
Different classes of ion channels have been implicated in sensing cold temperatures at mammalian ... more Different classes of ion channels have been implicated in sensing cold temperatures at mammalian thermoreceptor nerve endings. A major candidate is TRPM8, a non-selective cation channel of the transient receptor potential family, activated by menthol and low temperatures. We investigated the role of TRPM8 in cold sensing during transient expression in mouse cultured hippocampal neurones, a tissue that lacks endogenous expression of thermosensitive TRPs. In the absence of synaptic input, control hippocampal neurones were not excited by cooling. In contrast, all TRPM8-transfected hippocampal neurones were excited by cooling and menthol. However, in comparison to cold-sensitive trigeminal sensory neurones, hippocampal neurones exhibited much lower threshold temperatures, requiring temperatures below 27 • C to fire action potentials. These results directly demonstrate that expression of TRPM8 in mammalian neurones induces cold sensing, albeit at lower temperatures than native TRPM8-expressing neurones, suggesting the presence of additional modulatory mechanisms in the cold response of sensory neurones.
TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels... more TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels, is activated by voltage, low temperatures and cooling compounds. These properties and its restricted expression to small sensory neurons have made it the ion channel with the most advocated role in cold transduction. Recent work suggests that activation of TRPM8 by cold and menthol takes place through shifts in its voltage-activation curve, which cause the channel to open at physiological membrane potentials. By contrast, little is known about the actions of inhibitors on the function of TRPM8. We investigated the chemical and thermal modulation of TRPM8 in transfected HEK293 cells and in cold-sensitive primary sensory neurons. We show that cold-evoked TRPM8 responses are effectively suppressed by inhibitor compounds SKF96365, 4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide (BCTC) and 1,10-phenanthroline. These antagonists exert their effect by shifting the voltage dependence of TRPM8 activation towards more positive potentials. An opposite shift towards more negative potentials is achieved by the agonist menthol. Functionally, the bidirectional shift in channel gating translates into a change in the apparent temperature threshold of TRPM8-expressing cells. Accordingly, in the presence of the antagonist compounds, the apparent response-threshold temperature of TRPM8 is displaced towards colder temperatures, whereas menthol sensitizes the response, shifting the threshold in the opposite direction. Co-application of agonists and antagonists produces predictable cancellation of these effects, suggesting the convergence on a common molecular process. The potential for half maximal activation of TRPM8 activation by cold was ∼140 mV more negative in native channels compared to recombinant channels, with a much higher open probability at negative membrane potentials in the former. In functional terms, this difference translates into a shift in the apparent temperature threshold for activation towards higher temperatures for native currents. This difference in voltage-dependence readily explains the high threshold temperatures characteristic of many cold thermoreceptors. The modulation of TRPM8 activity by different chemical agents unveils an important flexibility in the temperature-response curve of TRPM8 channels and cold thermoreceptors.
Electrochemistry at Lipid Monolayer-Modified Liquid−Liquid Interfaces as an Improvement to Drug Partitioning Studies
The Journal of Physical Chemistry B, 2001
... Annika Mälkiä, Peter Liljeroth, Anna-Kaisa Kontturi, and Kyösti Kontturi*. Laboratory of Phys... more ... Annika Mälkiä, Peter Liljeroth, Anna-Kaisa Kontturi, and Kyösti Kontturi*. Laboratory of Physical Chemistry and Electrochemistry, Helsinki University of Technology, PO Box 6100, FIN-02015 HUT, Finland. J. Phys. Chem. B , 2001 ...
The hippocampus plays an important role in short term memory, learning and spatial navigation. A ... more The hippocampus plays an important role in short term memory, learning and spatial navigation. A characteristic feature of the hippocampal region is its expression of different electrical population rhythms and activities during different brain states. Physiological fluctuations in brain temperature affect the activity patterns in hippocampus, but the underlying cellular mechanisms are poorly understood. In this work, we investigated the thermal modulation of hippocampal activity at the cellular network level. Primary cell cultures of mouse E17 hippocampus displayed robust network activation upon light cooling of the extracellular solution from baseline physiological temperatures. The activity generated was dependent on action potential firing and excitatory glutamatergic synaptic transmission. Involvement of thermosensitive channels from the transient receptor potential (TRP) family in network activation by temperature changes was ruled out, whereas pharmacological and immunochemical experiments strongly pointed towards the involvement of temperature-sensitive two-pore-domain potassium channels (K 2P ), TREK/TRAAK family. In hippocampal slices we could show an increase in evoked and spontaneous synaptic activity produced by mild cooling in the physiological range that was prevented by chloroform, a K 2P channel opener. We propose that cold-induced closure of background TREK/TRAAK family channels increases the excitability of some hippocampal neurons, acting as a temperature-sensitive gate of network activation. Our findings in the hippocampus open the possibility that small temperature variations in the brain in vivo, associated with metabolism or blood flow oscillations, act as a switch mechanism of neuronal activity and determination of firing patterns through regulation of thermosensitive background potassium channel activity.
Treatment with resveratrol (RSV) has been shown to protect vulnerable neurons after various brain... more Treatment with resveratrol (RSV) has been shown to protect vulnerable neurons after various brain injuries and in neurodegenerative diseases. The mechanisms for the effects of RSV in brain are not fully understood, but RSV may affect the expression of various gene products. RSV is structurally related to the synthetic estrogen, diethylstilbestrol so the effects of RSV may be gender-specific. Here we studied the role of RSV in the regulation of dopamine transporter (DAT) in the striatum using male and female mice. The basic levels of DAT in the striatum showed no sex difference, but the levels increased significantly by RSV (20 mg/kg i.p.) in female but not in male mice. Pretreatment of mice with the selective estrogen receptor (ER), ERa-and ERb antagonist ICI 182,780, led to a complete block of RSV effect on DAT protein levels, suggesting that ERs are involved in the up-regulation of DAT by RSV. Similar data was also obtained in culture using human MESC2.10 and mouse SN4741 dopaminergic cells after treatment with RSV. Data further showed that RSV specifically induced gene transcription of DAT in the dopaminergic cells. These results show that estrogen receptors are involved in the up-regulation of DAT by RSV in the dopaminergic neurons, demonstrating a sex-dependent effect of RSV in the brain that may be of clinical importance.
Background: TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of t... more Background: TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of the transient receptor potential (TRP) ion channels. TRPM8 is activated by voltage, cold and cooling compounds such as menthol. Despite its essential role for cold temperature sensing in mammals, the pharmacology of TRPM8 is still in its infancy. Recently, tyrosine 745 (Y745) was identified as a critical residue for menthol sensitivity of the channel. In this report, we study the effect of mutating this residue on the action of several known TRPM8 antagonists: BCTC, capsazepine, SKF96365, and clotrimazole as well as two new inhibitor candidates, econazole and imidazole.
Polyelectrolyte/gold nanoparticle multilayers composed of poly(L-lysine) (pLys) and mercaptosucci... more Polyelectrolyte/gold nanoparticle multilayers composed of poly(L-lysine) (pLys) and mercaptosuccinic acid (MSA) stabilized gold nanoparticles (Au NPs) were built up using the electrostatic layer-by-layer self-assembly technique upon a gold electrode modified with a first layer of MSA. The assemblies were characterized using UV-vis absorption spectroscopy, cyclic and square-wave voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. Charge transport through the multilayer was studied experimentally as well as theoretically by using two different redox pairs [Fe(CN) 6 ] 3-/4and [Ru(NH 3 ) 6 ] 3+/2+ . This paper reports a large sensitivity to the charge of the outermost layer for the permeability of these assemblies to the probe ions. With the former redox pair, dramatic changes in the impedance response were obtained for thin multilayers each time a new layer was deposited. In the latter case, the multilayer behaves as a conductor exhibiting a strikingly lower impedance response, the electric current being enhanced as more layers are added for Au NP terminated multilayers. These results are interpreted quite satisfactorily by means of a capillary membrane model that encompasses the wide variety of behaviors observed. It is concluded that nonlinear slow diffusion through defects (pinholes) in the multilayer is the governing mechanism for the [Fe(CN) 6 ] 3-/4species, whereas electron transfer through the Au NPs is the dominant mechanism in the case of the [Ru(NH 3 ) 6 ] 3+/2+ pair.
Structurally related aminoacridine therapeutics (aminacrine, tacrine, velnacrine and proflavine) ... more Structurally related aminoacridine therapeutics (aminacrine, tacrine, velnacrine and proflavine) were studied electrochemically at the interface between two immiscible electrolyte solutions. To assess both their neutral and ionic partition coefficients, cyclic voltammetry was used to obtain the transfer potential as a function of the aqueous phase pH. Subsequently, in order to create a simple model of a biological membrane, a lipid monolayer was deposited at the liquid-liquid interface using the Langmuir-Blodgett technique. Electrochemical impedance spectroscopy was employed to study the interactions between the drug ions and the biomimetic phospholipid monolayer. Using a model incorporating a charge transfer mechanism coupled with potential dependent adsorption steps, the membrane activity of the studied drug ions could be estimated. The results indicate liquid-liquid electrochemistry to be a versatile tool for detecting differences in membrane activity even between structurally very similar drugs.
Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by ... more Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by cold temperature and cooling agents. TRPM8 is expressed in a subpopulation of cold-sensitive sensory neurons, as well as in the male urogenital system. TRPM8 is markedly upregulated in prostate cancer and in other tumors such as breast adenocarcinoma and melanoma. Moreover, recent studies suggest the potential involvement of TRPM8 channels in the pathophysiology of cold nociception and cold allodynia. This has led to a strong interest in the pursuit of novel modulators of TRPM8 channels. This review highlights our current knowledge of TRPM8 pharmacology and modulation mechanisms, detailing structural features important for TRPM8 gating by different agonists, the mechanism of antagonism by different compounds and the potential relevance of TRPM8 for treatment of various pathological conditions.
This thesis describes the development and characterisation of a novel biomembrane model suitable ... more This thesis describes the development and characterisation of a novel biomembrane model suitable for electrochemical investigation, and its applicability in studies of biologically related phenomena. The model system consists of a lipid monolayer, deposited at a polarisable liquid -liquid interface with the Langmuir -Blodgett technique. Combinations of ac and dc electrochemical techniques with theoretical models are employed to obtain information on the phase transfer and membrane interactions of charged therapeutics at the monolayer-covered interface. The thesis comprises six publications and a thorough introduction to biological and model membranes.
Membrane activity of biotechnological peptide drugs
Chemical Communications, 2003
Charged Langmuir-Blodgett monolayers deposited at an immobilised liquid-liquid interface have bee... more Charged Langmuir-Blodgett monolayers deposited at an immobilised liquid-liquid interface have been used as a simple model for a biological membrane to investigate the membrane activity of biotechnological oligopeptide drugs.
Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but little is k... more Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but little is known about the molecular mechanisms controlling these events. Peroxisome proliferator-activated receptor-gamma coactivator-1a (PGC-1a) is a transcriptional coactivator that is a master regulator of oxidative stress and mitochondrial metabolism. We show here that transgenic mice overexpressing PGC-1a in dopaminergic neurons are resistant against cell degeneration induced by the neurotoxin MPTP. The increase in neuronal viability was accompanied by elevated levels of mitochondrial antioxidants SOD2 and Trx2 in the substantia nigra of transgenic mice. PGC-1a overexpression also protected against MPTP-induced striatal loss of dopamine, and mitochondria from PGC-1a transgenic mice showed an increased respiratory control ratio compared with wild-type animals. To modulate PGC-1a, we employed the small molecular compound, resveratrol (RSV) that protected dopaminergic neurons against the MPTP-induced cell degeneration almost to the same extent as after PGC-1a overexpression. As studied in vitro, RSV activated PGC-1a in dopaminergic SN4741 cells via the deacetylase SIRT1, and enhanced PGC-1a gene transcription with increases in SOD2 and Trx2. Taken together, the results reveal an important function of PGC-1a in dopaminergic neurons to combat oxidative stress and increase neuronal viability. RSV and other compounds acting via SIRT1/PGC-1a may prove useful as neuroprotective agents in PD and possibly in other neurological disorders.
Uploads
Papers by Annika Malkia