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Madeline A. Shea - One of the best experts on this subject based on the ideXlab platform.

  • Backbone resonance assignments of complexes of human voltage-dependent Sodium Channel Nav1.2 IQ motif peptide bound to apo calmodulin and to the C-domain fragment of apo calmodulin.
    Biomolecular Nmr Assignments, 2017
    Co-Authors: Ryan Mahling, Adina M. Kilpatrick, Madeline A. Shea
    Abstract:

    Human voltage-gated Sodium Channel Nav1.2 has a single pore-forming α-subunit and two transmembrane β-subunits. Expressed primarily in the brain, Nav1.2 is critical for initiation and propagation of action potentials. Milliseconds after the pore opens, Sodium influx is terminated by inactivation processes mediated by regulatory proteins including calmodulin (CaM). Both calcium-free (apo) CaM and calcium-saturated CaM bind tightly to an IQ motif in the C-terminal tail of the α-subunit. Our thermodynamic studies and solution structure (2KXW) of a C-domain fragment of apo 13C,15N- CaM (CaMC) bound to an unlabeled peptide with the sequence of rat Nav1.2 IQ motif showed that apo CaMC (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaMC. However, we could not monitor the Nav1.2 residues directly, and no structure of full-length CaM (including the N-domain of CaM (CaMN)) was determined. To distinguish contributions of CaMN and CaMC, we used solution NMR spectroscopy to assign the backbone resonances of a complex containing a 13C,15N-labeled peptide with the sequence of human Nav1.2 IQ motif (Nav1.2IQp) bound to apo 13C,15N-CaM or apo 13C,15N-CaMC. Comparing the assignments of apo CaM in complex with Nav1.2IQp to those of free apo CaM showed that residues within CaMC were significantly perturbed, while residues within CaMN were essentially unchanged. The chemical shifts of residues in Nav1.2IQp and in the C-domain of CaM were nearly identical regardless of whether CaMN was covalently linked to CaMC. This suggests that CaMN does not influence apo CaM binding to Nav1.2IQp.

  • Recruitment of Calmodulin to the Tail of the Voltage-Gated Sodium Channel Nav1.2
    Biophysical Journal, 2015
    Co-Authors: Liam Hovey, Corinne Andresen, Dagan C. Marx, Madeline A. Shea
    Abstract:

    Voltage-gated Sodium Channels (NaV) found in excitable cells are responsible for the rising phase of action potentials. These multi-domain transmembrane proteins are regulated by calmodulin (CaM), a highly conserved eukaryotic protein that mediates many calcium-triggered signaling events. Inactivation of Sodium Channels depends on CaM-mediated feedback during repolarization. In the neuronal Sodium Channel Nav1.2, CaM binds at least two well-separated sites: an intracellular “inactivation” loop between domains DIII and DIV, and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail. The IQ motif is hypothesized to recruit calcium-free (apo) CaM, making it available to move to the III-IV linker after an influx of calcium. Despite a high degree of sequence identity, the equilibrium constants for CaM binding to nine human NaV IQ motifs span more than 3 orders of magnitude. Apo CaM binds to the Nav1.2 IQ motif with a dissociation constant (Kd) of ∼6 nM, while the Kd for binding the NaV1.9 IQ motif is ∼ 4 μM. Mutational analysis within the IQ motif has not been sufficient to explain the full range of CaM-binding affinities observed for human NaV sequences. Thus, we hypothesized that isoform-specific differences in upstream sequences were making energetically significant contributions to the free energy of binding CaM to Nav1.2. The roles of these residues are being investigated by monitoring CaM binding to biosensors containing mutant sequences of Sodium Channels bracketed by auto-fluorescent proteins YFP and CFP. Residue-specific information obtained by NMR will provide structural insight into the contributions of residues in the binding interface formed by NaV IQ motif sequences binding to calmodulin from multiple eukaryotes. NIH R01 GM57001.

  • Determinants of Preferential Binding of Apo Calmodulin to the IQ Motif of Neuronal Sodium Channel Nav1.2
    Biophysical Journal, 2014
    Co-Authors: Liam Hovey, Dagan C. Marx, Mark S. Miller, Jesse B. Yoder, Kristin M. Tefft, Madeline A. Shea
    Abstract:

    The neuronal voltage-gated Sodium Channel (Nav1.2) is regulated by calmodulin (CaM), a highly conserved, ubiquitous eukaryotic protein that mediates many calcium-triggered signaling events. Fast inactivation of the Channel depends on CaM-mediated feedback transduction of calcium flux during the repolarization phase of an action potential. CaM binds to an intracellular loop (the III-IV linker) and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail of the Channel. The Nav1.2 IQ motif binds only to the C-domain of CaM with high affinity to both its calcium-free (apo) and calcium-saturated states. However, the IQ motif binds more favorably to apo CaM than to calcium-saturated CaM. To determine the molecular basis for this calcium-dependent difference in association, mutational perturbations of residues in the Nav1.2 IQ motif were designed to disrupt close contacts observed in our solution (NMR) structure of the semi-open C-domain of apo-CaM bound to the IQ motif (2KXW.pdb). The contributions of these residues to binding energetics were determined by monitoring CaM-induced disruption of FRET in biosensors containing wild-type or mutant sequences of the IQ motif bracketed by auto-fluorescent proteins YFP and CFP. All mutations lowered affinity for calcium-saturated CaM, but they had uniformly more deleterious effects on the binding of apo CaM. Furthermore, the decrease in affinity for apo CaM caused by loss of the Ile-Gln pair was 30-fold greater than that observed for loss of the Tyr-Tyr pair. Thus, the energy of interaction between the Nav1.2 IQ motif and semi-open apo CaM is not accounted for primarily by the classical “aromatic anchors” that dominate interactions of calcium-saturated CaM with its target sequences in kinases, receptors and other Channels. Support: NIH R01 GM57001, Carver Charitable Trust Grant 01-224.

  • Calcium-Mediated Tailspin of Calmodulin on the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel Nav1.2
    Biophysical Journal, 2013
    Co-Authors: Madeline A. Shea, C. Andrew Fowler, Mark S. Miller, Jesse B. Yoder, Michael D. Feldkamp, Liping Yu
    Abstract:

    Nav1.2 is regulated by calmodulin (CaM), an essential calcium sensor with two homologous domains (N and C). CaM binds tightly to an IQ motif in the intracellular C-terminal tail of the pore-forming alpha subunit of Nav1.2 as well as to the inactivation gate. The IQ motif interacts with the “semi-open” cleft of apo CaM: I inserts into the cleft, while Q contacts the FG-turn of CaM (2KXW). To learn how CaM-IQ responds to increases in intracellular [Ca2+], we determined equilibrium constants for apo and calcium-saturated CaM binding to biosensors containing mutated IQ motif sequences sandwiched between YFP and CFP. Their quantum yields permit resolution of Kd values close to 1 nM from equilibrium titrations. Changes of Nav1.2 residues making close contacts with apo CaM were anticipated to diminish binding of both apo and calcium-saturated CaM. However, the quantitative effects differed by orders of magnitude. The affinity for calcium-saturated CaM dropped by factors of 10-100, while effects on apo CaM binding were more severe. Thus, the CaM-IQ interface differs dramatically depending on calcium-saturation of CaM. NMR studies of a complex of (Ca2+)2-CaM-C-domain bound to the IQ motif showed that calcium binding opens CaM but also causes it to pivot by 180° so that it binds to the IQ motif in the opposite direction. The I of the IQ motif contacts the “open” hydrophobic cleft of (Ca2+)2-CaM, but the Q points towards the linker between the N- and C-domains of CaM. In conjunction with 2KXW, this new structure provides the first pair of high resolution structures for apo and calcium-saturated CaM bound to a single IQ motif. Support: NIH R01 GM57001 and Carver Charitable Trust Grant 01-224.

  • Calmodulin Regulation of the Neuronal Voltage-Dependent Sodium Channel
    Biophysical Journal, 2010
    Co-Authors: Michael D. Feldkamp, Liping Yu, Madeline A. Shea
    Abstract:

    Calmodulin (CaM) is an essential eukaryotic calcium sensor comprised of two homologous domains (N, C). Ca2+ binding to CaM changes its conformation and determines how CaM recognizes and regulates target proteins such as the neuronal voltage-dependent Sodium Channel (Nav1.2) which is essential for the generation and propagation of action potentials. Nav1.2 is a multimer with one pore-forming α-subunit and one or more β-subunits. CaM binds to an IQ-motif (IQxxxBGxxxB, B=K,R) of Nav1.2 that is near the C-terminus of the α-subunit. Prior thermodynamic studies showed that this IQ peptide (Nav1.2IQp, KRKQEEVSAIVIQRAYRRYLLKQKVKK) selectively lowers the Ca2+-binding affinity sites in the C-domain of CaM, without affecting the N-domain (Theoharis et al, Biochemistry 2008). This selective decrease correlates with Nav1.2IQp having a higher affinity for apo CaM than for calcium-saturated CaM. Structural studies of complexes of CaM bound to target peptides or proteins demonstrated that the 4-helix bundle of the CaM C-domain adopts an “open” conformation when Ca2+-saturated. There is only one high-resolution structure (2IX7) of apo CaM bound to an IQ motif; it shows the C-domain having a “semi-open” conformation. To understand the Ca2+-dependent conformational switching CaM when regulating Nav1.2, we applied heteronuclear NMR methods. Amide exchange, hnNOE, and chemical shift perturbation experiments revealed residue-specific changes consistent with a “semi-open” conformation of the apo C-domain of CaM when bound to Nav1.2IQp. NMR experiments are complete and analysis is underway to determine the solution structure of the apo C-domain of CaM bound to Nav1.2IQp. Understanding the interface between CaM and the IQ-motif of the Channel will result in a more complete model of how CaM regulates Nav1.2 function at low physiological [Ca2+] in neuronal tissues. NIH GM57001

Christopher M. Flores - One of the best experts on this subject based on the ideXlab platform.

  • Validation of a Patch Clamp Screening Protocol That Simultaneously Measures Compound Activity in Multiple States of the Voltage-Gated Sodium Channel Nav1.2
    Assay and Drug Development Technologies, 2011
    Co-Authors: Edward Beck, Christopher M. Flores
    Abstract:

    Abstract Hyperactivity of voltage-gated Sodium Channels underlies, at least in part, a range of pathological states, including pain and epilepsy. Selective blockers of these Channels may offer effective treatment of such disorders. Currently employed methods to screen for Sodium Channel blockers, however, are inadequate to rationally identify mechanistically diverse blockers, limiting the potential range of indications that may be treated by such agents. Here, we describe an improved patch clamp screening assay that increases the mechanistic diversity of Sodium Channel blockers being identified. Using QPatch HT, a medium-throughput, automated patch clamp system, we tested three common Sodium Channel blockers (phenytoin, lidocaine, and tetrodotoxin) with distinct mechanistic profiles at Nav1.2. The single-voltage protocol employed in this assay simultaneously measured the compound activity in multiple states, including the slow inactivated state, of the Channel. A long compound incubation period (10 s) was...

  • Inhibition of the rat brain Sodium Channel Nav1.2 after prolonged exposure to gabapentin
    Epilepsy Research, 2006
    Co-Authors: Tasha L. Reitz, Yan Wang, Christopher M. Flores
    Abstract:

    Abstract Prolonged exposure of neurons to gabapentin inhibits repetitive firing of Na+-dependent action potentials. Here, we studied the effect of such prolonged exposure to gabapentin on a rat Sodium Channel, Nav1.2. After 3 days of continuous incubation with gabapentin (10–1000 μM), Nav1.2 current density was decreased dose-dependently relative to untreated cells. The reduction was 57% at 30 μM gabapentin, while higher concentrations (100–1000 μM) did not result in greater effects. Prolonged treatment with gabapentin also caused the Channel to inactivate at more hyperpolarized potentials. These effects provide a mechanistic basis for the inhibition of Na+-dependent repetitive firing upon prolonged exposure to gabapentin and may contribute to its anticonvulsant activity.

  • Short communication Inhibition of the rat brain Sodium Channel Nav1.2 after prolonged exposure to gabapentin
    2006
    Co-Authors: Tasha L. Reitz, Yan Wang, Christopher M. Flores
    Abstract:

    Prolonged exposure of neurons to gabapentin inhibits repetitive firing of Na + -dependent action potentials. Here, we studied the effect of such prolonged exposure to gabapentin on a rat Sodium Channel, Nav1.2. After 3 days of continuous incubation with gabapentin (10–1000M), Nav1.2 current density was decreased dose-dependently relative to untreated cells. The reduction was 57% at 30M gabapentin, while higher concentrations (100–1000M) did not result in greater effects. Prolonged treatment with gabapentin also caused the Channel to inactivate at more hyperpolarized potentials. These effects provide a mechanistic basis for the inhibition of Na + -dependent repetitive firing upon prolonged exposure to gabapentin and may contribute to its anticonvulsant activity.

Liam Hovey - One of the best experts on this subject based on the ideXlab platform.

  • Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent Sodium Channel Nav1.2 ☆
    Biophysical Chemistry, 2017
    Co-Authors: Liam Hovey, Dagan C. Marx, C. Andrew Fowler, Ryan Mahling, Mark S. Miller, Jesse B. Yoder, Kristin M. Tefft, Brett C. Waite
    Abstract:

    Abstract Several members of the voltage-gated Sodium Channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated Sodium Channel Nav1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat Nav1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~ 3 nM) and (Ca2 +)4-CaM (~ 85 nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca2 +)4-CaM, NMR demonstrated that Nav1.2 IQ motif peptide (Nav1.2IQp) exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2 +)2-CaMC bound to Nav1.2IQp. The polarity of (Ca2 +)2-CaMC relative to the IQ motif was opposite to that seen in apo CaMC-Nav1.2IQp (2KXW), revealing that CaMC recognizes nested, anti-parallel sites in Nav1.2IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaMN allowing interactions with non-IQ Nav1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.

  • Recruitment of Calmodulin to the Tail of the Voltage-Gated Sodium Channel Nav1.2
    Biophysical Journal, 2015
    Co-Authors: Liam Hovey, Corinne Andresen, Dagan C. Marx, Madeline A. Shea
    Abstract:

    Voltage-gated Sodium Channels (NaV) found in excitable cells are responsible for the rising phase of action potentials. These multi-domain transmembrane proteins are regulated by calmodulin (CaM), a highly conserved eukaryotic protein that mediates many calcium-triggered signaling events. Inactivation of Sodium Channels depends on CaM-mediated feedback during repolarization. In the neuronal Sodium Channel Nav1.2, CaM binds at least two well-separated sites: an intracellular “inactivation” loop between domains DIII and DIV, and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail. The IQ motif is hypothesized to recruit calcium-free (apo) CaM, making it available to move to the III-IV linker after an influx of calcium. Despite a high degree of sequence identity, the equilibrium constants for CaM binding to nine human NaV IQ motifs span more than 3 orders of magnitude. Apo CaM binds to the Nav1.2 IQ motif with a dissociation constant (Kd) of ∼6 nM, while the Kd for binding the NaV1.9 IQ motif is ∼ 4 μM. Mutational analysis within the IQ motif has not been sufficient to explain the full range of CaM-binding affinities observed for human NaV sequences. Thus, we hypothesized that isoform-specific differences in upstream sequences were making energetically significant contributions to the free energy of binding CaM to Nav1.2. The roles of these residues are being investigated by monitoring CaM binding to biosensors containing mutant sequences of Sodium Channels bracketed by auto-fluorescent proteins YFP and CFP. Residue-specific information obtained by NMR will provide structural insight into the contributions of residues in the binding interface formed by NaV IQ motif sequences binding to calmodulin from multiple eukaryotes. NIH R01 GM57001.

  • Determinants of Preferential Binding of Apo Calmodulin to the IQ Motif of Neuronal Sodium Channel Nav1.2
    Biophysical Journal, 2014
    Co-Authors: Liam Hovey, Dagan C. Marx, Mark S. Miller, Jesse B. Yoder, Kristin M. Tefft, Madeline A. Shea
    Abstract:

    The neuronal voltage-gated Sodium Channel (Nav1.2) is regulated by calmodulin (CaM), a highly conserved, ubiquitous eukaryotic protein that mediates many calcium-triggered signaling events. Fast inactivation of the Channel depends on CaM-mediated feedback transduction of calcium flux during the repolarization phase of an action potential. CaM binds to an intracellular loop (the III-IV linker) and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail of the Channel. The Nav1.2 IQ motif binds only to the C-domain of CaM with high affinity to both its calcium-free (apo) and calcium-saturated states. However, the IQ motif binds more favorably to apo CaM than to calcium-saturated CaM. To determine the molecular basis for this calcium-dependent difference in association, mutational perturbations of residues in the Nav1.2 IQ motif were designed to disrupt close contacts observed in our solution (NMR) structure of the semi-open C-domain of apo-CaM bound to the IQ motif (2KXW.pdb). The contributions of these residues to binding energetics were determined by monitoring CaM-induced disruption of FRET in biosensors containing wild-type or mutant sequences of the IQ motif bracketed by auto-fluorescent proteins YFP and CFP. All mutations lowered affinity for calcium-saturated CaM, but they had uniformly more deleterious effects on the binding of apo CaM. Furthermore, the decrease in affinity for apo CaM caused by loss of the Ile-Gln pair was 30-fold greater than that observed for loss of the Tyr-Tyr pair. Thus, the energy of interaction between the Nav1.2 IQ motif and semi-open apo CaM is not accounted for primarily by the classical “aromatic anchors” that dominate interactions of calcium-saturated CaM with its target sequences in kinases, receptors and other Channels. Support: NIH R01 GM57001, Carver Charitable Trust Grant 01-224.

Tasha L. Reitz - One of the best experts on this subject based on the ideXlab platform.

  • Inhibition of the rat brain Sodium Channel Nav1.2 after prolonged exposure to gabapentin
    Epilepsy Research, 2006
    Co-Authors: Tasha L. Reitz, Yan Wang, Christopher M. Flores
    Abstract:

    Abstract Prolonged exposure of neurons to gabapentin inhibits repetitive firing of Na+-dependent action potentials. Here, we studied the effect of such prolonged exposure to gabapentin on a rat Sodium Channel, Nav1.2. After 3 days of continuous incubation with gabapentin (10–1000 μM), Nav1.2 current density was decreased dose-dependently relative to untreated cells. The reduction was 57% at 30 μM gabapentin, while higher concentrations (100–1000 μM) did not result in greater effects. Prolonged treatment with gabapentin also caused the Channel to inactivate at more hyperpolarized potentials. These effects provide a mechanistic basis for the inhibition of Na+-dependent repetitive firing upon prolonged exposure to gabapentin and may contribute to its anticonvulsant activity.

  • Short communication Inhibition of the rat brain Sodium Channel Nav1.2 after prolonged exposure to gabapentin
    2006
    Co-Authors: Tasha L. Reitz, Yan Wang, Christopher M. Flores
    Abstract:

    Prolonged exposure of neurons to gabapentin inhibits repetitive firing of Na + -dependent action potentials. Here, we studied the effect of such prolonged exposure to gabapentin on a rat Sodium Channel, Nav1.2. After 3 days of continuous incubation with gabapentin (10–1000M), Nav1.2 current density was decreased dose-dependently relative to untreated cells. The reduction was 57% at 30M gabapentin, while higher concentrations (100–1000M) did not result in greater effects. Prolonged treatment with gabapentin also caused the Channel to inactivate at more hyperpolarized potentials. These effects provide a mechanistic basis for the inhibition of Na + -dependent repetitive firing upon prolonged exposure to gabapentin and may contribute to its anticonvulsant activity.

Liping Yu - One of the best experts on this subject based on the ideXlab platform.

  • Calcium-Mediated Tailspin of Calmodulin on the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel Nav1.2
    Biophysical Journal, 2013
    Co-Authors: Madeline A. Shea, C. Andrew Fowler, Mark S. Miller, Jesse B. Yoder, Michael D. Feldkamp, Liping Yu
    Abstract:

    Nav1.2 is regulated by calmodulin (CaM), an essential calcium sensor with two homologous domains (N and C). CaM binds tightly to an IQ motif in the intracellular C-terminal tail of the pore-forming alpha subunit of Nav1.2 as well as to the inactivation gate. The IQ motif interacts with the “semi-open” cleft of apo CaM: I inserts into the cleft, while Q contacts the FG-turn of CaM (2KXW). To learn how CaM-IQ responds to increases in intracellular [Ca2+], we determined equilibrium constants for apo and calcium-saturated CaM binding to biosensors containing mutated IQ motif sequences sandwiched between YFP and CFP. Their quantum yields permit resolution of Kd values close to 1 nM from equilibrium titrations. Changes of Nav1.2 residues making close contacts with apo CaM were anticipated to diminish binding of both apo and calcium-saturated CaM. However, the quantitative effects differed by orders of magnitude. The affinity for calcium-saturated CaM dropped by factors of 10-100, while effects on apo CaM binding were more severe. Thus, the CaM-IQ interface differs dramatically depending on calcium-saturation of CaM. NMR studies of a complex of (Ca2+)2-CaM-C-domain bound to the IQ motif showed that calcium binding opens CaM but also causes it to pivot by 180° so that it binds to the IQ motif in the opposite direction. The I of the IQ motif contacts the “open” hydrophobic cleft of (Ca2+)2-CaM, but the Q points towards the linker between the N- and C-domains of CaM. In conjunction with 2KXW, this new structure provides the first pair of high resolution structures for apo and calcium-saturated CaM bound to a single IQ motif. Support: NIH R01 GM57001 and Carver Charitable Trust Grant 01-224.

  • Calmodulin Regulation of the Neuronal Voltage-Dependent Sodium Channel
    Biophysical Journal, 2010
    Co-Authors: Michael D. Feldkamp, Liping Yu, Madeline A. Shea
    Abstract:

    Calmodulin (CaM) is an essential eukaryotic calcium sensor comprised of two homologous domains (N, C). Ca2+ binding to CaM changes its conformation and determines how CaM recognizes and regulates target proteins such as the neuronal voltage-dependent Sodium Channel (Nav1.2) which is essential for the generation and propagation of action potentials. Nav1.2 is a multimer with one pore-forming α-subunit and one or more β-subunits. CaM binds to an IQ-motif (IQxxxBGxxxB, B=K,R) of Nav1.2 that is near the C-terminus of the α-subunit. Prior thermodynamic studies showed that this IQ peptide (Nav1.2IQp, KRKQEEVSAIVIQRAYRRYLLKQKVKK) selectively lowers the Ca2+-binding affinity sites in the C-domain of CaM, without affecting the N-domain (Theoharis et al, Biochemistry 2008). This selective decrease correlates with Nav1.2IQp having a higher affinity for apo CaM than for calcium-saturated CaM. Structural studies of complexes of CaM bound to target peptides or proteins demonstrated that the 4-helix bundle of the CaM C-domain adopts an “open” conformation when Ca2+-saturated. There is only one high-resolution structure (2IX7) of apo CaM bound to an IQ motif; it shows the C-domain having a “semi-open” conformation. To understand the Ca2+-dependent conformational switching CaM when regulating Nav1.2, we applied heteronuclear NMR methods. Amide exchange, hnNOE, and chemical shift perturbation experiments revealed residue-specific changes consistent with a “semi-open” conformation of the apo C-domain of CaM when bound to Nav1.2IQp. NMR experiments are complete and analysis is underway to determine the solution structure of the apo C-domain of CaM bound to Nav1.2IQp. Understanding the interface between CaM and the IQ-motif of the Channel will result in a more complete model of how CaM regulates Nav1.2 function at low physiological [Ca2+] in neuronal tissues. NIH GM57001