In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn... more In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn locus in the CFTR (cystic fibrosis transmembrane conductance regulator) gene is a frequent disease mutation with incomplete penetrance. This T5 allele will result in a high proportion of CFTR transcripts that lack exon 9, whose translation products will not contribute to apical chloride channel activity. Besides the polymorphic Tn locus, more than 120 polymorphisms have been described in the CFTR gene. We hypothesized that the combination of particular alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. Analysis of three polymorphic loci with frequent alleles in the general population showed that, in addition to the known effect of the Tn locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci: (TG)m and M470V. On a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, and (TG)12 gave a sixfold increase, compared with the (TG)10 allele. T5 CFTR genes derived from patients were found to carry a high number of TG repeats, while T5 CFTR genes derived from healthy CF fathers harbored a low number of TG repeats. Moreover, it was found that M470 CFTR proteins matured more slowly, and that they had a 1.7-fold increased intrinsic chloride channel activity compared with V470 CFTR proteins, suggesting that the M470V locus might also play a role in the partial penetrance of T5 as a disease mutation. Such polyvariant mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations, and be of relevance in other genetic diseases.
Ion channels are pore-forming transmembrane proteins that allow ions to permeate biological membr... more Ion channels are pore-forming transmembrane proteins that allow ions to permeate biological membranes. Pore structure plays a crucial role in determining the ion permeation and selectivity properties of particular channels. In the past few decades, efforts have been undertaken to identify key elements of the pore regions of different classes of ion channels. In this review, we summarize current knowledge about permeation and selectivity of channel proteins from the transient receptor potential (TRP) superfamily. Whereas all TRP channels are permeable for cations, only two TRP channels are impermeable for Ca2+ (TRPM4, TRPM5), and two others are highly Ca2+ permeable (TRPV5, TRPV6). Despite the great advances in the TRP channel field during the past decade, only a limited number of reports have dealt with functional characterization of pore properties, biophysical aspects of cation permeation, or description of pore structures of TRP channels. This review gives an overview of available experimental and theoretical data and discusses the functional impact of pore-structure modifications on TRP channel properties.
TRPV4 is a widely expressed cation channel of the `transient receptor potential' (TRP) family tha... more TRPV4 is a widely expressed cation channel of the `transient receptor potential' (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4αPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5',6'-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.
Pflugers Archiv-european Journal of Physiology, 1993
We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca2+-adenosine ... more We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca2+-adenosine 5′-triphosphatase (Ca2+-ATPase), on membrane currents and on the intracellular Ca2+ concentration ([Ca2+]i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patchclamp technique in the whole-cell mode and [Ca2+]i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 μmol/l induced a slow increase in [Ca2+]i to a peak value of 400±110 nmol/l above a resting level of 120±35 nmol/l, and then slowly declined to a new steady-state level of 315±90 nmol/l (n=33). The thapsigargin-induced increase in [Ca2+]i depended on the extracellular Ca2+ concentration ([Ca2+]o: it declined after removal of extracellular Ca2+, but increased again when [Ca2+]o was augmented, indicating that the response depends on a transmembrane influx of Ca2+ ions. The peak amplitude of the histamine-induced Ca2+ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca2+]i induced by thapsigargin than during the rising phase of the changes in [Ca2+]i. The decline of the Ca2+ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant τ equal to 24.5±5 s (n=7). In the presence of thapsigargin, the decline was much slower (n =8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast τ value of 29.4±4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of −40 mV. Voltage ramps applied before and during the development of this current indicated that a non-selective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca2+ transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca2+ pump in endothelial cells increases [Ca2+]i. The tonic component of this increase might be partly due to opening of non-selective Ca2+-permeable cation channels activated by depletion of intracellular stores.
The functional impact of ion channels in vascular endothelial cells (ECs) is still a matter of co... more The functional impact of ion channels in vascular endothelial cells (ECs) is still a matter of controversy. This review describes different types of ion channels in ECs and their role in electrogenesis, Ca2+ signaling, vessel permeability, cell-cell communication, mechano-sensor functions, and pH and volume regulation. One major function of ion channels in ECs is the control of Ca2+ influx either by a direct modulation of the Ca2+ influx pathway or by indirect modulation of K+ and Cl- channels, thereby clamping the membrane at a sufficiently negative potential to provide the necessary driving force for a sustained Ca2+ influx. We discuss various mechanisms of Ca2+ influx stimulation: those that activate nonselective, Ca(2+)-permeable cation channels or those that activate Ca(2+)-selective channels, exclusively or partially operated by the filling state of intracellular Ca2+ stores. We also describe the role of various Ca(2+)- and shear stress-activated K+ channels and different types of Cl- channels for the regulation of the membrane potential.
TRPV4 is a widely expressed cation channel of the &am... more TRPV4 is a widely expressed cation channel of the 'transient receptor potential' (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4alphaPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5',6'-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.
Pflugers Archiv-european Journal of Physiology, 1995
Volume-activated Cl− currents (ICl,vol) and cell growth have been measured in cultured endothelia... more Volume-activated Cl− currents (ICl,vol) and cell growth have been measured in cultured endothelial cells from bovine pulmonary artery (CPAE) in the absence and presence of compounds which block these currents. The anti-oestrogen drug tamoxifen, which efficiently arrests the growth of breast cancer cells (l), inhibits both ICl,vol and cell proliferation with IC50 of 3.8 and 4.8 μmol/l respectively. NPPB and quinine, which also block ICl,vol, inhibit the growth of CPAE cells as well. Current and cell growth were closely correlated under all these conditions. We conclude that ICl,vol might be involved in the control of endothelial cell growth and thus might be important for the modulation of vascularisation and vascular remodelling.
We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential a... more We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i.Mibefradil (10 μM) hyperpolarized the membrane potential of CPAE cells from its mean control value of −26.6±0.6 mV (n=7) to −59.8±1.7 mV (n=6). A depolarizing effect was observed at higher concentrations (−13.7±0.6 mV, n=4, 30 μM mibefradil).Mibefradil inhibited Ca2+-activated Cl− currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki=4.7±0.18 μM, n=8).Mibefradil also inhibited volume-sensitive Cl− currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki=5.4±0.22 μM, n=6).The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 μM.Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6±6.5% (n=8) in the presence of 10 μM mibefradil.Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7±0.12 μM (n=3), which is similar to the concentration for half-maximal block of Cl− channels.These actions of mibefradil on Cl− channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i.Mibefradil (10 μM) hyperpolarized the membrane potential of CPAE cells from its mean control value of −26.6±0.6 mV (n=7) to −59.8±1.7 mV (n=6). A depolarizing effect was observed at higher concentrations (−13.7±0.6 mV, n=4, 30 μM mibefradil).Mibefradil inhibited Ca2+-activated Cl− currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki=4.7±0.18 μM, n=8).Mibefradil also inhibited volume-sensitive Cl− currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki=5.4±0.22 μM, n=6).The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 μM.Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6±6.5% (n=8) in the presence of 10 μM mibefradil.Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7±0.12 μM (n=3), which is similar to the concentration for half-maximal block of Cl− channels.These actions of mibefradil on Cl− channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.
In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn... more In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn locus in the CFTR (cystic fibrosis transmembrane conductance regulator) gene is a frequent disease mutation with incomplete penetrance. This T5 allele will result in a high proportion of CFTR transcripts that lack exon 9, whose translation products will not contribute to apical chloride channel activity. Besides the polymorphic Tn locus, more than 120 polymorphisms have been described in the CFTR gene. We hypothesized that the combination of particular alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. Analysis of three polymorphic loci with frequent alleles in the general population showed that, in addition to the known effect of the Tn locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci: (TG)m and M470V. On a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, and (TG)12 gave a sixfold increase, compared with the (TG)10 allele. T5 CFTR genes derived from patients were found to carry a high number of TG repeats, while T5 CFTR genes derived from healthy CF fathers harbored a low number of TG repeats. Moreover, it was found that M470 CFTR proteins matured more slowly, and that they had a 1.7-fold increased intrinsic chloride channel activity compared with V470 CFTR proteins, suggesting that the M470V locus might also play a role in the partial penetrance of T5 as a disease mutation. Such polyvariant mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations, and be of relevance in other genetic diseases.
Ion channels are pore-forming transmembrane proteins that allow ions to permeate biological membr... more Ion channels are pore-forming transmembrane proteins that allow ions to permeate biological membranes. Pore structure plays a crucial role in determining the ion permeation and selectivity properties of particular channels. In the past few decades, efforts have been undertaken to identify key elements of the pore regions of different classes of ion channels. In this review, we summarize current knowledge about permeation and selectivity of channel proteins from the transient receptor potential (TRP) superfamily. Whereas all TRP channels are permeable for cations, only two TRP channels are impermeable for Ca2+ (TRPM4, TRPM5), and two others are highly Ca2+ permeable (TRPV5, TRPV6). Despite the great advances in the TRP channel field during the past decade, only a limited number of reports have dealt with functional characterization of pore properties, biophysical aspects of cation permeation, or description of pore structures of TRP channels. This review gives an overview of available experimental and theoretical data and discusses the functional impact of pore-structure modifications on TRP channel properties.
TRPV4 is a widely expressed cation channel of the `transient receptor potential' (TRP) family tha... more TRPV4 is a widely expressed cation channel of the `transient receptor potential' (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4αPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5',6'-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.
Pflugers Archiv-european Journal of Physiology, 1993
We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca2+-adenosine ... more We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca2+-adenosine 5′-triphosphatase (Ca2+-ATPase), on membrane currents and on the intracellular Ca2+ concentration ([Ca2+]i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patchclamp technique in the whole-cell mode and [Ca2+]i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 μmol/l induced a slow increase in [Ca2+]i to a peak value of 400±110 nmol/l above a resting level of 120±35 nmol/l, and then slowly declined to a new steady-state level of 315±90 nmol/l (n=33). The thapsigargin-induced increase in [Ca2+]i depended on the extracellular Ca2+ concentration ([Ca2+]o: it declined after removal of extracellular Ca2+, but increased again when [Ca2+]o was augmented, indicating that the response depends on a transmembrane influx of Ca2+ ions. The peak amplitude of the histamine-induced Ca2+ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca2+]i induced by thapsigargin than during the rising phase of the changes in [Ca2+]i. The decline of the Ca2+ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant τ equal to 24.5±5 s (n=7). In the presence of thapsigargin, the decline was much slower (n =8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast τ value of 29.4±4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of −40 mV. Voltage ramps applied before and during the development of this current indicated that a non-selective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca2+ transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca2+ pump in endothelial cells increases [Ca2+]i. The tonic component of this increase might be partly due to opening of non-selective Ca2+-permeable cation channels activated by depletion of intracellular stores.
The functional impact of ion channels in vascular endothelial cells (ECs) is still a matter of co... more The functional impact of ion channels in vascular endothelial cells (ECs) is still a matter of controversy. This review describes different types of ion channels in ECs and their role in electrogenesis, Ca2+ signaling, vessel permeability, cell-cell communication, mechano-sensor functions, and pH and volume regulation. One major function of ion channels in ECs is the control of Ca2+ influx either by a direct modulation of the Ca2+ influx pathway or by indirect modulation of K+ and Cl- channels, thereby clamping the membrane at a sufficiently negative potential to provide the necessary driving force for a sustained Ca2+ influx. We discuss various mechanisms of Ca2+ influx stimulation: those that activate nonselective, Ca(2+)-permeable cation channels or those that activate Ca(2+)-selective channels, exclusively or partially operated by the filling state of intracellular Ca2+ stores. We also describe the role of various Ca(2+)- and shear stress-activated K+ channels and different types of Cl- channels for the regulation of the membrane potential.
TRPV4 is a widely expressed cation channel of the &am... more TRPV4 is a widely expressed cation channel of the 'transient receptor potential' (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4alphaPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5',6'-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.
Pflugers Archiv-european Journal of Physiology, 1995
Volume-activated Cl− currents (ICl,vol) and cell growth have been measured in cultured endothelia... more Volume-activated Cl− currents (ICl,vol) and cell growth have been measured in cultured endothelial cells from bovine pulmonary artery (CPAE) in the absence and presence of compounds which block these currents. The anti-oestrogen drug tamoxifen, which efficiently arrests the growth of breast cancer cells (l), inhibits both ICl,vol and cell proliferation with IC50 of 3.8 and 4.8 μmol/l respectively. NPPB and quinine, which also block ICl,vol, inhibit the growth of CPAE cells as well. Current and cell growth were closely correlated under all these conditions. We conclude that ICl,vol might be involved in the control of endothelial cell growth and thus might be important for the modulation of vascularisation and vascular remodelling.
We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential a... more We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i.Mibefradil (10 μM) hyperpolarized the membrane potential of CPAE cells from its mean control value of −26.6±0.6 mV (n=7) to −59.8±1.7 mV (n=6). A depolarizing effect was observed at higher concentrations (−13.7±0.6 mV, n=4, 30 μM mibefradil).Mibefradil inhibited Ca2+-activated Cl− currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki=4.7±0.18 μM, n=8).Mibefradil also inhibited volume-sensitive Cl− currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki=5.4±0.22 μM, n=6).The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 μM.Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6±6.5% (n=8) in the presence of 10 μM mibefradil.Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7±0.12 μM (n=3), which is similar to the concentration for half-maximal block of Cl− channels.These actions of mibefradil on Cl− channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i.Mibefradil (10 μM) hyperpolarized the membrane potential of CPAE cells from its mean control value of −26.6±0.6 mV (n=7) to −59.8±1.7 mV (n=6). A depolarizing effect was observed at higher concentrations (−13.7±0.6 mV, n=4, 30 μM mibefradil).Mibefradil inhibited Ca2+-activated Cl− currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki=4.7±0.18 μM, n=8).Mibefradil also inhibited volume-sensitive Cl− currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki=5.4±0.22 μM, n=6).The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 μM.Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6±6.5% (n=8) in the presence of 10 μM mibefradil.Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7±0.12 μM (n=3), which is similar to the concentration for half-maximal block of Cl− channels.These actions of mibefradil on Cl− channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.
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Papers by Bernd Nilius