The Experts below are selected from a list of 78825 Experts worldwide ranked by ideXlab platform
Bernhard E Flucher - One of the best experts on this subject based on the ideXlab platform.
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current modulation and membrane targeting of the calcium channel α1c subunit are independent functions of the β subunit
The Journal of Physiology, 1999Co-Authors: Uli Gerster, Birgit Neuhuber, Klaus Groschner, Jorg Striessnig, Bernhard E FlucherAbstract:The β subunits of voltage-sensitive calcium channels facilitate the incorporation of channels into the plasma membrane and modulate calcium currents. In order to determine whether these two effects of the β subunit are interdependent or independent of each other we studied plasma membrane incorporation of the channel subunits with green fluorescent protein and immunofluorescence labelling, and current modulation with whole-cell and single-channel patch-clamp recordings in transiently transfected human embryonic kidney tsA201 cells. Coexpression of rabbit cardiac muscle α1C with rabbit skeletal muscle β1a, rabbit heart/brain β2a or rat brain β3 subunits resulted in the colocalization of α1C with β and in a marked translocation of the channel complexes into the plasma membrane. In parallel, the whole-cell current density and single-channel open probability were increased. Furthermore, the β2a isoform specifically altered the voltage dependence of current activation and the inactivation kinetics. A single amino acid substitution in the β subunit Interaction domain of α1C (α1CY467S) disrupted the colocalization and plasma membrane targeting of both subunits without affecting the β subunit-induced modulation of whole-cell currents and single-channel properties. These results show that the modulation of calcium currents by β subunits can be explained by β subunit-induced changes of single-channel properties, but the formation of stable α1C-β complexes and their increased incorporation into the plasma membrane appear not to be necessary for functional modulation. Voltage-sensitive calcium channels are multimeric protein complexes formed by the α1 subunit and the auxiliary subunits α2δ, β and γ (Leung et al. 1987; Takahashi et al. 1987; Vaghy et al. 1987). The α1 subunit by itself shows the characteristic properties of a voltage-gated ion channel, i.e. voltage sensing, ion permeation and drug binding. The physiological roles of the auxiliary subunits are currently the subject of intensive investigations. The β subunit modulates calcium currents by increasing the current density and by changing the current kinetics when coexpressed in heterologous expression systems (Lacerda et al. 1991; Varadi et al. 1991; Lory et al. 1992). Furthermore, it has been suggested that the β subunit is involved in the targeting of the α1 subunit to the plasma membrane (Chien et al. 1995; Gregg et al. 1996). Both the α1 and β subunits exist in multiple tissue-specific isoforms, which differ from one another in their primary structure and in their functional properties (Birnbaumer et al. 1994; Isom et al. 1994). The skeletal muscle α1S isoform, for example, shows slow activation and inactivation kinetics compared with the other α1 isoforms. Also, whereas α1C expressed in heterologous expression systems exhibits currents even in the absence of auxiliary subunits (Perez-Garcia et al. 1995), expression of α1S in heterologous systems rarely gives rise to measurable calcium currents (Johnson et al. 1997). The β subunit isoforms differ in their current modulation (Hullin et al. 1992; Sather et al. 1993; Parent et al. 1997) and, when expressed alone, in their subcellular distribution (Chien et al. 1995, 1996; Brice et al. 1997). For example, β2a drastically reduced the speed of inactivation when coexpressed with the neuronal α1E subunit in oocytes (Parent et al. 1997), whereas other β subunit isoforms showed only minor effects on current inactivation. Analogously, β2a differs from most other β isoforms in that it was localized in the plasma membrane when expressed without an α1 subunit in a heterologous expression system (Chien et al. 1995), whereas β1a, β3 and β4 showed a cytoplasmic localization (Brice et al. 1997; Neuhuber et al. 1998b). A conserved β subunit binding motif has been identified in the cytoplasmic loop between repeats I and II of α1S, α1A, α1B and α1C (Pragnell et al. 1994). Point mutations within this binding motif perturbed α1-β binding and affected calcium current properties when the neuronal α1A isoform was coexpressed with β1b in oocytes. A point mutation (Y366S) within the β subunit binding motif in the I-II linker of the skeletal muscle α1S resulted in the expected loss of α1S-β1a binding, but the probability that tsA201 human embryonic kidney cells cotransfected with α1SY366S and β1a exhibited calcium currents was still increased by the β1a subunit (Neuhuber et al. 1998b). This indicates that stable binding of β1a to the known motif in the I-II linker of α1S is not necessary for the β subunit to increase the frequency of current expression. Thus, association of β with this Interaction domain in the cytoplasmic I-II linker plays an important role in β subunit-dependent modulation of calcium currents, but other mechanisms for α1-β Interaction may exist. De Waard et al. (1994, 1996) identified a conserved 30 amino acid domain in the β subunit that is complementary to the binding site in the I-II linker on α1A and is involved in the Interaction with this subunit. Mutations within this domain of β perturbed binding to α1A and affected modulation of calcium current properties. However, this domain in the β subunit cannot account for all observed modulatory effects of α1A-β Interactions, since certain truncated β subunits were only affected in their modulation of inactivation kinetics, not in current stimulation. Therefore, a region of the β subunit other than that interacting with the I-II linker of α1 may be involved in the modulation of α1A (De Waard et al. 1994). Moreover, using chimeras of different β isoforms Olcese et al. (1994) and Qin et al. (1996) have found that regulation of activation and inactivation of α1E channels are two separable functions of the β subunit, suggesting the existence of two Separate Interaction domains on each of the subunits. Indeed, Tareilus et al. (1997) identified a second β subunit binding domain within the last 277 amino acids of the C-terminus of α1E, and Walker et al. (1998) identified a low affinity binding site in the carboxy-terminal region of α1A that accounts for β4-induced modulation of current inactivation. Despite this progress in understanding the function of the calcium channel β subunit the mechanism of current modulation by β remains largely unresolved. For example, it is still controversial whether increased insertion of channels into the plasma membrane and modulation of current properties are two independent functions of the β subunit or whether the latter is a direct result of the former. Also, we do not know whether α1 and β form a stable complex in which β serves as a necessary cofactor or mediator of modulatory signals, or whether association and dissociation of the β subunit in itself is the modulatory mechanism. To address these questions we studied the Interactions of three different β subunit isoforms with α1C and an α1C mutant with a single amino acid substitution in the β subunit Interaction domain of the I-II linker (α1CY467S) using a combination of structural and functional techniques. This approach allowed us to distinguish β isoform-specific effects from common effects of α1C-β Interactions and to demonstrate that increased membrane incorporation and modulation of channel properties are two independent effects of β subunits. Single-channel analysis showed that changes in the open probability are sufficiently large to explain the increase in whole-cell current density observed upon coexpression of the β subunit. Further, the comparison of β subunit effects on wild-type and mutant α1C shows that this increase in current density occurs even without the formation of stable α1C-β complexes or their increased incorporation into the plasma membrane.
Gerry Stahl - One of the best experts on this subject based on the ideXlab platform.
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the integration of synchronous communication across dual Interaction spaces
Computer Supported Collaborative Learning, 2007Co-Authors: Martin Muhlpfordt, Gerry StahlAbstract:Dual Interaction spaces--that combine text chat with a shared graphical work area--have been developed in recent years as CSCL applications to support the synchronous construction and discussion of shared artifacts by distributed small groups of students. However, the simple juxtaposition of the two spaces raises numerous issues for users: How can objects in the shared workspace be referenced from within the chat? How can users track and comprehend all the various simultaneous activities? How can participants coordinate their multifaceted actions? We present three steps toward integration of activities across Separate Interaction spaces: support for deictic references, implementation of a history feature and display of social awareness information.
Uli Gerster - One of the best experts on this subject based on the ideXlab platform.
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current modulation and membrane targeting of the calcium channel α1c subunit are independent functions of the β subunit
The Journal of Physiology, 1999Co-Authors: Uli Gerster, Birgit Neuhuber, Klaus Groschner, Jorg Striessnig, Bernhard E FlucherAbstract:The β subunits of voltage-sensitive calcium channels facilitate the incorporation of channels into the plasma membrane and modulate calcium currents. In order to determine whether these two effects of the β subunit are interdependent or independent of each other we studied plasma membrane incorporation of the channel subunits with green fluorescent protein and immunofluorescence labelling, and current modulation with whole-cell and single-channel patch-clamp recordings in transiently transfected human embryonic kidney tsA201 cells. Coexpression of rabbit cardiac muscle α1C with rabbit skeletal muscle β1a, rabbit heart/brain β2a or rat brain β3 subunits resulted in the colocalization of α1C with β and in a marked translocation of the channel complexes into the plasma membrane. In parallel, the whole-cell current density and single-channel open probability were increased. Furthermore, the β2a isoform specifically altered the voltage dependence of current activation and the inactivation kinetics. A single amino acid substitution in the β subunit Interaction domain of α1C (α1CY467S) disrupted the colocalization and plasma membrane targeting of both subunits without affecting the β subunit-induced modulation of whole-cell currents and single-channel properties. These results show that the modulation of calcium currents by β subunits can be explained by β subunit-induced changes of single-channel properties, but the formation of stable α1C-β complexes and their increased incorporation into the plasma membrane appear not to be necessary for functional modulation. Voltage-sensitive calcium channels are multimeric protein complexes formed by the α1 subunit and the auxiliary subunits α2δ, β and γ (Leung et al. 1987; Takahashi et al. 1987; Vaghy et al. 1987). The α1 subunit by itself shows the characteristic properties of a voltage-gated ion channel, i.e. voltage sensing, ion permeation and drug binding. The physiological roles of the auxiliary subunits are currently the subject of intensive investigations. The β subunit modulates calcium currents by increasing the current density and by changing the current kinetics when coexpressed in heterologous expression systems (Lacerda et al. 1991; Varadi et al. 1991; Lory et al. 1992). Furthermore, it has been suggested that the β subunit is involved in the targeting of the α1 subunit to the plasma membrane (Chien et al. 1995; Gregg et al. 1996). Both the α1 and β subunits exist in multiple tissue-specific isoforms, which differ from one another in their primary structure and in their functional properties (Birnbaumer et al. 1994; Isom et al. 1994). The skeletal muscle α1S isoform, for example, shows slow activation and inactivation kinetics compared with the other α1 isoforms. Also, whereas α1C expressed in heterologous expression systems exhibits currents even in the absence of auxiliary subunits (Perez-Garcia et al. 1995), expression of α1S in heterologous systems rarely gives rise to measurable calcium currents (Johnson et al. 1997). The β subunit isoforms differ in their current modulation (Hullin et al. 1992; Sather et al. 1993; Parent et al. 1997) and, when expressed alone, in their subcellular distribution (Chien et al. 1995, 1996; Brice et al. 1997). For example, β2a drastically reduced the speed of inactivation when coexpressed with the neuronal α1E subunit in oocytes (Parent et al. 1997), whereas other β subunit isoforms showed only minor effects on current inactivation. Analogously, β2a differs from most other β isoforms in that it was localized in the plasma membrane when expressed without an α1 subunit in a heterologous expression system (Chien et al. 1995), whereas β1a, β3 and β4 showed a cytoplasmic localization (Brice et al. 1997; Neuhuber et al. 1998b). A conserved β subunit binding motif has been identified in the cytoplasmic loop between repeats I and II of α1S, α1A, α1B and α1C (Pragnell et al. 1994). Point mutations within this binding motif perturbed α1-β binding and affected calcium current properties when the neuronal α1A isoform was coexpressed with β1b in oocytes. A point mutation (Y366S) within the β subunit binding motif in the I-II linker of the skeletal muscle α1S resulted in the expected loss of α1S-β1a binding, but the probability that tsA201 human embryonic kidney cells cotransfected with α1SY366S and β1a exhibited calcium currents was still increased by the β1a subunit (Neuhuber et al. 1998b). This indicates that stable binding of β1a to the known motif in the I-II linker of α1S is not necessary for the β subunit to increase the frequency of current expression. Thus, association of β with this Interaction domain in the cytoplasmic I-II linker plays an important role in β subunit-dependent modulation of calcium currents, but other mechanisms for α1-β Interaction may exist. De Waard et al. (1994, 1996) identified a conserved 30 amino acid domain in the β subunit that is complementary to the binding site in the I-II linker on α1A and is involved in the Interaction with this subunit. Mutations within this domain of β perturbed binding to α1A and affected modulation of calcium current properties. However, this domain in the β subunit cannot account for all observed modulatory effects of α1A-β Interactions, since certain truncated β subunits were only affected in their modulation of inactivation kinetics, not in current stimulation. Therefore, a region of the β subunit other than that interacting with the I-II linker of α1 may be involved in the modulation of α1A (De Waard et al. 1994). Moreover, using chimeras of different β isoforms Olcese et al. (1994) and Qin et al. (1996) have found that regulation of activation and inactivation of α1E channels are two separable functions of the β subunit, suggesting the existence of two Separate Interaction domains on each of the subunits. Indeed, Tareilus et al. (1997) identified a second β subunit binding domain within the last 277 amino acids of the C-terminus of α1E, and Walker et al. (1998) identified a low affinity binding site in the carboxy-terminal region of α1A that accounts for β4-induced modulation of current inactivation. Despite this progress in understanding the function of the calcium channel β subunit the mechanism of current modulation by β remains largely unresolved. For example, it is still controversial whether increased insertion of channels into the plasma membrane and modulation of current properties are two independent functions of the β subunit or whether the latter is a direct result of the former. Also, we do not know whether α1 and β form a stable complex in which β serves as a necessary cofactor or mediator of modulatory signals, or whether association and dissociation of the β subunit in itself is the modulatory mechanism. To address these questions we studied the Interactions of three different β subunit isoforms with α1C and an α1C mutant with a single amino acid substitution in the β subunit Interaction domain of the I-II linker (α1CY467S) using a combination of structural and functional techniques. This approach allowed us to distinguish β isoform-specific effects from common effects of α1C-β Interactions and to demonstrate that increased membrane incorporation and modulation of channel properties are two independent effects of β subunits. Single-channel analysis showed that changes in the open probability are sufficiently large to explain the increase in whole-cell current density observed upon coexpression of the β subunit. Further, the comparison of β subunit effects on wild-type and mutant α1C shows that this increase in current density occurs even without the formation of stable α1C-β complexes or their increased incorporation into the plasma membrane.
Martin Muhlpfordt - One of the best experts on this subject based on the ideXlab platform.
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the integration of synchronous communication across dual Interaction spaces
Computer Supported Collaborative Learning, 2007Co-Authors: Martin Muhlpfordt, Gerry StahlAbstract:Dual Interaction spaces--that combine text chat with a shared graphical work area--have been developed in recent years as CSCL applications to support the synchronous construction and discussion of shared artifacts by distributed small groups of students. However, the simple juxtaposition of the two spaces raises numerous issues for users: How can objects in the shared workspace be referenced from within the chat? How can users track and comprehend all the various simultaneous activities? How can participants coordinate their multifaceted actions? We present three steps toward integration of activities across Separate Interaction spaces: support for deictic references, implementation of a history feature and display of social awareness information.
Jorg Striessnig - One of the best experts on this subject based on the ideXlab platform.
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current modulation and membrane targeting of the calcium channel α1c subunit are independent functions of the β subunit
The Journal of Physiology, 1999Co-Authors: Uli Gerster, Birgit Neuhuber, Klaus Groschner, Jorg Striessnig, Bernhard E FlucherAbstract:The β subunits of voltage-sensitive calcium channels facilitate the incorporation of channels into the plasma membrane and modulate calcium currents. In order to determine whether these two effects of the β subunit are interdependent or independent of each other we studied plasma membrane incorporation of the channel subunits with green fluorescent protein and immunofluorescence labelling, and current modulation with whole-cell and single-channel patch-clamp recordings in transiently transfected human embryonic kidney tsA201 cells. Coexpression of rabbit cardiac muscle α1C with rabbit skeletal muscle β1a, rabbit heart/brain β2a or rat brain β3 subunits resulted in the colocalization of α1C with β and in a marked translocation of the channel complexes into the plasma membrane. In parallel, the whole-cell current density and single-channel open probability were increased. Furthermore, the β2a isoform specifically altered the voltage dependence of current activation and the inactivation kinetics. A single amino acid substitution in the β subunit Interaction domain of α1C (α1CY467S) disrupted the colocalization and plasma membrane targeting of both subunits without affecting the β subunit-induced modulation of whole-cell currents and single-channel properties. These results show that the modulation of calcium currents by β subunits can be explained by β subunit-induced changes of single-channel properties, but the formation of stable α1C-β complexes and their increased incorporation into the plasma membrane appear not to be necessary for functional modulation. Voltage-sensitive calcium channels are multimeric protein complexes formed by the α1 subunit and the auxiliary subunits α2δ, β and γ (Leung et al. 1987; Takahashi et al. 1987; Vaghy et al. 1987). The α1 subunit by itself shows the characteristic properties of a voltage-gated ion channel, i.e. voltage sensing, ion permeation and drug binding. The physiological roles of the auxiliary subunits are currently the subject of intensive investigations. The β subunit modulates calcium currents by increasing the current density and by changing the current kinetics when coexpressed in heterologous expression systems (Lacerda et al. 1991; Varadi et al. 1991; Lory et al. 1992). Furthermore, it has been suggested that the β subunit is involved in the targeting of the α1 subunit to the plasma membrane (Chien et al. 1995; Gregg et al. 1996). Both the α1 and β subunits exist in multiple tissue-specific isoforms, which differ from one another in their primary structure and in their functional properties (Birnbaumer et al. 1994; Isom et al. 1994). The skeletal muscle α1S isoform, for example, shows slow activation and inactivation kinetics compared with the other α1 isoforms. Also, whereas α1C expressed in heterologous expression systems exhibits currents even in the absence of auxiliary subunits (Perez-Garcia et al. 1995), expression of α1S in heterologous systems rarely gives rise to measurable calcium currents (Johnson et al. 1997). The β subunit isoforms differ in their current modulation (Hullin et al. 1992; Sather et al. 1993; Parent et al. 1997) and, when expressed alone, in their subcellular distribution (Chien et al. 1995, 1996; Brice et al. 1997). For example, β2a drastically reduced the speed of inactivation when coexpressed with the neuronal α1E subunit in oocytes (Parent et al. 1997), whereas other β subunit isoforms showed only minor effects on current inactivation. Analogously, β2a differs from most other β isoforms in that it was localized in the plasma membrane when expressed without an α1 subunit in a heterologous expression system (Chien et al. 1995), whereas β1a, β3 and β4 showed a cytoplasmic localization (Brice et al. 1997; Neuhuber et al. 1998b). A conserved β subunit binding motif has been identified in the cytoplasmic loop between repeats I and II of α1S, α1A, α1B and α1C (Pragnell et al. 1994). Point mutations within this binding motif perturbed α1-β binding and affected calcium current properties when the neuronal α1A isoform was coexpressed with β1b in oocytes. A point mutation (Y366S) within the β subunit binding motif in the I-II linker of the skeletal muscle α1S resulted in the expected loss of α1S-β1a binding, but the probability that tsA201 human embryonic kidney cells cotransfected with α1SY366S and β1a exhibited calcium currents was still increased by the β1a subunit (Neuhuber et al. 1998b). This indicates that stable binding of β1a to the known motif in the I-II linker of α1S is not necessary for the β subunit to increase the frequency of current expression. Thus, association of β with this Interaction domain in the cytoplasmic I-II linker plays an important role in β subunit-dependent modulation of calcium currents, but other mechanisms for α1-β Interaction may exist. De Waard et al. (1994, 1996) identified a conserved 30 amino acid domain in the β subunit that is complementary to the binding site in the I-II linker on α1A and is involved in the Interaction with this subunit. Mutations within this domain of β perturbed binding to α1A and affected modulation of calcium current properties. However, this domain in the β subunit cannot account for all observed modulatory effects of α1A-β Interactions, since certain truncated β subunits were only affected in their modulation of inactivation kinetics, not in current stimulation. Therefore, a region of the β subunit other than that interacting with the I-II linker of α1 may be involved in the modulation of α1A (De Waard et al. 1994). Moreover, using chimeras of different β isoforms Olcese et al. (1994) and Qin et al. (1996) have found that regulation of activation and inactivation of α1E channels are two separable functions of the β subunit, suggesting the existence of two Separate Interaction domains on each of the subunits. Indeed, Tareilus et al. (1997) identified a second β subunit binding domain within the last 277 amino acids of the C-terminus of α1E, and Walker et al. (1998) identified a low affinity binding site in the carboxy-terminal region of α1A that accounts for β4-induced modulation of current inactivation. Despite this progress in understanding the function of the calcium channel β subunit the mechanism of current modulation by β remains largely unresolved. For example, it is still controversial whether increased insertion of channels into the plasma membrane and modulation of current properties are two independent functions of the β subunit or whether the latter is a direct result of the former. Also, we do not know whether α1 and β form a stable complex in which β serves as a necessary cofactor or mediator of modulatory signals, or whether association and dissociation of the β subunit in itself is the modulatory mechanism. To address these questions we studied the Interactions of three different β subunit isoforms with α1C and an α1C mutant with a single amino acid substitution in the β subunit Interaction domain of the I-II linker (α1CY467S) using a combination of structural and functional techniques. This approach allowed us to distinguish β isoform-specific effects from common effects of α1C-β Interactions and to demonstrate that increased membrane incorporation and modulation of channel properties are two independent effects of β subunits. Single-channel analysis showed that changes in the open probability are sufficiently large to explain the increase in whole-cell current density observed upon coexpression of the β subunit. Further, the comparison of β subunit effects on wild-type and mutant α1C shows that this increase in current density occurs even without the formation of stable α1C-β complexes or their increased incorporation into the plasma membrane.
