Paul Kammermeier - Academia.edu (original) (raw)
Papers by Paul Kammermeier
Biophysical Journal, 2018
likelihood, and allowing the model to shrink and expand dynamically, we use the data itself to le... more likelihood, and allowing the model to shrink and expand dynamically, we use the data itself to learn about the system and uncover properties or dynamics that would be hidden by other methods. This method offers a flexible and simple way to allow the data to dictate the appropriate model structure instead of having the experimenter impose a rigid structure beforehand. Here we first validate this method by determining (a) the number of diffusive populations, (b) the average diffusion coefficient for each term, (c) the population weight fraction for each term, and (d) the rates of conversion from one term to another in simulations of heterogeneous diffusion. We then extend our investigations to simple experimental systems, and finally we apply our algorithm to determine the model and dynamics of the membrane-bound keystone virulence protein TcpP in live Vibrio cholerae cells.
Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases i... more Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several x-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here we describe the coarse topography of the cASIC1 intracellular domains determined by FRET, measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 Å and that the N and proximal segment of the C tail from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments towards the plasma membrane. Taken together, our study furnishes a coarse topographic map of the ASIC intracellular domains while providing directionality and context to intracellular conformational changes induced by extracellular acidification.
<b>Copyright information:</b>Taken from "Surface clustering of metabotropic glut... more <b>Copyright information:</b>Taken from "Surface clustering of metabotropic glutamate receptor 1 induced by long Homer proteins"BMC Neuroscience 2006;7():1-1.Published online 4 Jan 2006PMCID:PMC1361788.Copyright © 2006 Kammermeier; licensee BioMed Central Ltd. Two representative neurons from each group are shown. The total number of neurons examined for each group were: mGluR1-GFP (26 cells), + Homer 1a (9), + Homer 1b (12), + Homer 1c (12), + Homer 2b (34), + Homer 3 (7).
<b>Copyright information:</b>Taken from "Surface clustering of metabotropic glut... more <b>Copyright information:</b>Taken from "Surface clustering of metabotropic glutamate receptor 1 induced by long Homer proteins"BMC Neuroscience 2006;7():1-1.Published online 4 Jan 2006PMCID:PMC1361788.Copyright © 2006 Kammermeier; licensee BioMed Central Ltd. Two representative neurons from each group are shown. The total number of neurons examined for each group were: mGluR1-GFP (19 cells), + Homer 1a (5), + Homer 1b (6), + Homer 1c (9), + Homer 2b (8), + Homer 3 (10).
olpharm.aspetjournals.org D ow nloaded from 2 Running title: Functional interaction of mGluR1 and... more olpharm.aspetjournals.org D ow nloaded from 2 Running title: Functional interaction of mGluR1 and mGluR5.
eLife, 2021
Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases i... more Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several X-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here, we describe the coarse topography of the chicken ASIC1 intracellular domains determined by fluorescence resonance energy transfer (FRET), measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 Å and that the N and C tails from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments toward...
Neuropharmacology, 2021
Metabotropic glutamate receptors (mGluRs) are an essential component of the mammalian central ner... more Metabotropic glutamate receptors (mGluRs) are an essential component of the mammalian central nervous system. These receptors modulate neuronal excitability in response to extracellular glutamate through the activation of intracellular heterotrimeric G proteins. Like most other class C G protein-coupled receptors, mGluRs function as obligate dimer proteins, meaning they need to form dimer complexes before becoming functional receptors. All mGluRs possess the ability to homodimerize, but studies over the past ten years have demonstrated these receptors are also capable of forming heterodimers in specific patterns. These mGluR heterodimers appear to have their own unique biophysical behavior and pharmacology with both native and synthetic compounds with few rules having been identified that allow for prediction of the consequences of any particular mGluR pair forming heterodimers. Here, we review the relevant literature demonstrating the existence and consequences of mGluR heterodimerization. By collecting biophysical and pharmacological data of several mGluR heterodimers we demonstrate the lack of generalizable behavior of these complexes indicating that each individual dimeric pair needs to be investigated independently. Additionally, by combining sequence alignment and structural analysis, we propose that interactions between the β4-A Helix Loop and the D Helix in the extracellular domain of these receptors are the structural components that dictate heterodimerization compatibility. Finally, we discuss the potential implications of mGluR heterodimerization from the viewpoints of further developing our understanding of neuronal physiology and leveraging mGluRs as a therapeutic target for the treatment of pathophysiology.
Biophysical Journal, 2021
Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra- ... more Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra- and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. W...
Pharmacology Research & Perspectives, 2019
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Journal of Neurophysiology, 1998
Kammermeier, Paul J. and Stephen W. Jones. Facilitation of L-type calcium current in thalamic neu... more Kammermeier, Paul J. and Stephen W. Jones. Facilitation of L-type calcium current in thalamic neurons. J. Neurophysiol. 79: 410–417, 1998. We have studied facilitation of the L-type calcium current in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus. Currents were recorded after pretreatment with 1 μM ω-conotoxin GVIA and 5 μM ω-conotoxin MVIIC, to better isolate L-current. Long, strong depolarizations induced slow tail currents at negative voltages, but did not affect currents at voltages where channels were strongly activated. The initial peak tail current was not measurably increased. The time course of recovery from facilitation paralleled the time course of the tail current, indicating that facilitation does not outlast channel closing. The kinase inhibitors staurosporine and H-7 and the phosphatase inhibitor okadaic acid had no significant effect on L-current facilitation compared with control, but facilitation was greater with H-7 than with okadaic ac...
The Journal of Neuroscience, 2000
Calcium current modulation by the muscarinic cholinergic agonist oxotremorine methiodide (oxo-M) ... more Calcium current modulation by the muscarinic cholinergic agonist oxotremorine methiodide (oxo-M) was examined in sympathetic neurons from the superior cervical ganglion of the rat. Oxo-M strongly inhibited calcium currents via voltage-dependent (VD) and voltage-independent (VI) pathways. These pathways could be separated with the use of the specific M 1 acetylcholine receptor antagonist M 1-toxin and with pertussis toxin (PTX) treatment. Expression by nuclear cDNA injection of the regulator of G-protein signaling (RGS2) or a phospholipase C1 C-terminal construct (PLC-ct) selectively reduced VI oxo-M modulation in PTX-treated and untreated cells. Expression of the G␥ buffers transducin (G␣ tr) and a G-protein-coupled-receptor kinase (GRK3) construct (MAS-GRK3) eliminated oxo-M modulation. Activation of the heterologously expressed neurokinin type 1 receptor, a G␣ q/11-coupled receptor, resulted in VI calcium current modulation. This modulation was eliminated with coexpression of G␣ tr or MAS-GRK3. Cells expressing G 1 ␥ 2 were tonically inhibited via the VD pathway. Application of oxo-M to these cells produced VI modulation and reduced the amount of current inhibited via the VD pathway. Together, these results confirm the requirement for G␥ in VD modulation and implicate G␣ q-GTP and G␥ as components in the potentially novel VI pathway.
Journal of Neurophysiology, 1997
Kammermeier, Paul J. and Stephen W. Jones. High-voltage-activated calcium currents in neurons acu... more Kammermeier, Paul J. and Stephen W. Jones. High-voltage-activated calcium currents in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus. J. Neurophysiol. 77: 465–475, 1997. We studied the high-voltage-activated (HVA) calcium currents in cells isolated from the ventrobasal nucleus of the rat thalamus with the use of the whole cell patch-clamp technique. Low-voltage-activated current was inactivated by the use of long voltage steps or 100-ms prepulses to −20 mV. We used channel blocking agents to characterize the currents that make up the HVA current. The dihydropyridine (DHP) antagonist nimodipine (5 μM) reversibly blocked 33 ± 1% (mean ± SE), and ω-conotoxin GVIA (1 μM) irreversibly blocked 25 ± 5%. The current resistant to DHPs and ω-conotoxin GVIA was inhibited almost completely by ω-conotoxin MVIIC (90 ± 5% at 3–5 μM) and was partially inhibited by ω-agatoxin IVA (54 ± 4% block at 1 μM). We conclude that there are at least four main HVA currents in thalami...
The Journal of Neuroscience, 2000
Group I metabotropic glutamate receptors (mGluR1 and 5) couple to intracellular calcium pools by ... more Group I metabotropic glutamate receptors (mGluR1 and 5) couple to intracellular calcium pools by a family of proteins, termed Homer, that cross-link the receptor to inositol trisphosphate receptors. mGluRs also couple to membrane ion channels via G-proteins. The role of Homer proteins in channel modulation was investigated by expressing mGluRs and various forms of Homer in rat superior cervical ganglion (SCG) sympathetic neurons by intranuclear cDNA injection. Expression of cross-linkingcapable forms of Homer (Homer 1b, 1c, 2, and 3, termed long forms) occluded group I mGluR-mediated N-type calcium and M-type potassium current modulation. This effect was specific for group I mGluRs. mGluR2 (group II)-mediated inhibition of N-channels was unaltered. Long forms of Homer decreased modulation of N-and M-type currents but did not selectively block distinct G-protein pathways. Short forms of Homer, which cannot self-multimerize (Homer 1a and a Homer 2 C-terminal deletion), did not alter mGluR-ion channel coupling. When coexpressed with long forms of Homer, short forms restored the mGluR1a-mediated calcium current modulation in an apparent dose-dependent manner. Homer 2b induced cell surface clusters of mGluR5 in SCG neurons. Conversely, a uniform distribution was observed when mGluR5 was expressed alone or with Homer short forms. These studies indicate that long and short forms of Homer compete for binding to mGluRs and regulate their coupling to ion channels. In vivo, the immediate early Homer 1a is anticipated to enhance ion channel modulation and to disrupt coupling to releasable intracellular calcium pools. Thus, Homer may regulate the magnitude and predominate signaling output of group I mGluRs.
Cold Spring Harbor protocols, Jan 5, 2017
From patch-clamp techniques to recombinant DNA technologies, three-dimensional protein modeling, ... more From patch-clamp techniques to recombinant DNA technologies, three-dimensional protein modeling, and optogenetics, diverse and sophisticated methods have been used to study ion channels and how they determine the electrical properties of cells.
Channels (Austin, Tex.), Jan 24, 2017
Type two voltage gated calcium (CaV2) channels are the primary mediators of neurotransmission at ... more Type two voltage gated calcium (CaV2) channels are the primary mediators of neurotransmission at neuronal presynapses, but their function at neural soma is also important in regulating excitability (1) . Mechanisms that regulate CaV2 channel expression at synapses have been studied extensively, which motivated us to perform similar studies in the soma. Rat sympathetic neurons from the superior cervical ganglion (SCG) natively express CaV2.2 and CaV2.3 (2) . We noted previously that heterologous expression of CaV2.1 but not CaV2.2 results in increased calcium current in SCG neurons (3) . In the present study, we extended these observations to show that both CaV2.1 and CaV2.3 expression resulted in increased calcium currents while CaV2.2 expression did not. Further, CaV2.1 could displace native CaV2.2 channels, but CaV2.3 expression could not. Heterologous expression of the individual accessory subunits α2δ-1, α2δ-2, α2δ-3, or β4 alone failed to increase current density, suggesting th...
The Journal of pharmacology and experimental therapeutics, 2017
In rat sympathetic neurons from the superior cervical ganglia (SCG) expressing metabotropic gluta... more In rat sympathetic neurons from the superior cervical ganglia (SCG) expressing metabotropic glutamate receptor mGluR1 or mGluR5, overexpression of scaffolding Homer proteins, which bind to a Homer ligand in their C termini, cause receptor clustering and uncoupling from ion channel modulation. In the absence of recombinant Homer protein overexpression, uncoupling of mGluRs from voltage-dependent channels can be induced by expression of Preso1, an adaptor of proline-directed kinases that phosphorylates the Homer ligand and recruits binding of endogenous Homer proteins. Here we show that in SCG neurons expressing mGluR1 and the tyrosine receptor kinase B, treatment with brain-derived neurotrophic factor (BDNF) produces a similar uncoupling of the receptors from calcium channels. We investigated the pathways that mediate this uncoupling and compared it with uncoupling observed with Preso1 expression. Both BDNF- and Preso1-induced uncoupling require residues T1151 and S1154 in the mGluR1...
Biophysical Journal, 2018
likelihood, and allowing the model to shrink and expand dynamically, we use the data itself to le... more likelihood, and allowing the model to shrink and expand dynamically, we use the data itself to learn about the system and uncover properties or dynamics that would be hidden by other methods. This method offers a flexible and simple way to allow the data to dictate the appropriate model structure instead of having the experimenter impose a rigid structure beforehand. Here we first validate this method by determining (a) the number of diffusive populations, (b) the average diffusion coefficient for each term, (c) the population weight fraction for each term, and (d) the rates of conversion from one term to another in simulations of heterogeneous diffusion. We then extend our investigations to simple experimental systems, and finally we apply our algorithm to determine the model and dynamics of the membrane-bound keystone virulence protein TcpP in live Vibrio cholerae cells.
Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases i... more Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several x-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here we describe the coarse topography of the cASIC1 intracellular domains determined by FRET, measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 Å and that the N and proximal segment of the C tail from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments towards the plasma membrane. Taken together, our study furnishes a coarse topographic map of the ASIC intracellular domains while providing directionality and context to intracellular conformational changes induced by extracellular acidification.
<b>Copyright information:</b>Taken from "Surface clustering of metabotropic glut... more <b>Copyright information:</b>Taken from "Surface clustering of metabotropic glutamate receptor 1 induced by long Homer proteins"BMC Neuroscience 2006;7():1-1.Published online 4 Jan 2006PMCID:PMC1361788.Copyright © 2006 Kammermeier; licensee BioMed Central Ltd. Two representative neurons from each group are shown. The total number of neurons examined for each group were: mGluR1-GFP (26 cells), + Homer 1a (9), + Homer 1b (12), + Homer 1c (12), + Homer 2b (34), + Homer 3 (7).
<b>Copyright information:</b>Taken from "Surface clustering of metabotropic glut... more <b>Copyright information:</b>Taken from "Surface clustering of metabotropic glutamate receptor 1 induced by long Homer proteins"BMC Neuroscience 2006;7():1-1.Published online 4 Jan 2006PMCID:PMC1361788.Copyright © 2006 Kammermeier; licensee BioMed Central Ltd. Two representative neurons from each group are shown. The total number of neurons examined for each group were: mGluR1-GFP (19 cells), + Homer 1a (5), + Homer 1b (6), + Homer 1c (9), + Homer 2b (8), + Homer 3 (10).
olpharm.aspetjournals.org D ow nloaded from 2 Running title: Functional interaction of mGluR1 and... more olpharm.aspetjournals.org D ow nloaded from 2 Running title: Functional interaction of mGluR1 and mGluR5.
eLife, 2021
Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases i... more Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several X-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here, we describe the coarse topography of the chicken ASIC1 intracellular domains determined by fluorescence resonance energy transfer (FRET), measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 Å and that the N and C tails from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments toward...
Neuropharmacology, 2021
Metabotropic glutamate receptors (mGluRs) are an essential component of the mammalian central ner... more Metabotropic glutamate receptors (mGluRs) are an essential component of the mammalian central nervous system. These receptors modulate neuronal excitability in response to extracellular glutamate through the activation of intracellular heterotrimeric G proteins. Like most other class C G protein-coupled receptors, mGluRs function as obligate dimer proteins, meaning they need to form dimer complexes before becoming functional receptors. All mGluRs possess the ability to homodimerize, but studies over the past ten years have demonstrated these receptors are also capable of forming heterodimers in specific patterns. These mGluR heterodimers appear to have their own unique biophysical behavior and pharmacology with both native and synthetic compounds with few rules having been identified that allow for prediction of the consequences of any particular mGluR pair forming heterodimers. Here, we review the relevant literature demonstrating the existence and consequences of mGluR heterodimerization. By collecting biophysical and pharmacological data of several mGluR heterodimers we demonstrate the lack of generalizable behavior of these complexes indicating that each individual dimeric pair needs to be investigated independently. Additionally, by combining sequence alignment and structural analysis, we propose that interactions between the β4-A Helix Loop and the D Helix in the extracellular domain of these receptors are the structural components that dictate heterodimerization compatibility. Finally, we discuss the potential implications of mGluR heterodimerization from the viewpoints of further developing our understanding of neuronal physiology and leveraging mGluRs as a therapeutic target for the treatment of pathophysiology.
Biophysical Journal, 2021
Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra- ... more Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra- and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. W...
Pharmacology Research & Perspectives, 2019
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Journal of Neurophysiology, 1998
Kammermeier, Paul J. and Stephen W. Jones. Facilitation of L-type calcium current in thalamic neu... more Kammermeier, Paul J. and Stephen W. Jones. Facilitation of L-type calcium current in thalamic neurons. J. Neurophysiol. 79: 410–417, 1998. We have studied facilitation of the L-type calcium current in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus. Currents were recorded after pretreatment with 1 μM ω-conotoxin GVIA and 5 μM ω-conotoxin MVIIC, to better isolate L-current. Long, strong depolarizations induced slow tail currents at negative voltages, but did not affect currents at voltages where channels were strongly activated. The initial peak tail current was not measurably increased. The time course of recovery from facilitation paralleled the time course of the tail current, indicating that facilitation does not outlast channel closing. The kinase inhibitors staurosporine and H-7 and the phosphatase inhibitor okadaic acid had no significant effect on L-current facilitation compared with control, but facilitation was greater with H-7 than with okadaic ac...
The Journal of Neuroscience, 2000
Calcium current modulation by the muscarinic cholinergic agonist oxotremorine methiodide (oxo-M) ... more Calcium current modulation by the muscarinic cholinergic agonist oxotremorine methiodide (oxo-M) was examined in sympathetic neurons from the superior cervical ganglion of the rat. Oxo-M strongly inhibited calcium currents via voltage-dependent (VD) and voltage-independent (VI) pathways. These pathways could be separated with the use of the specific M 1 acetylcholine receptor antagonist M 1-toxin and with pertussis toxin (PTX) treatment. Expression by nuclear cDNA injection of the regulator of G-protein signaling (RGS2) or a phospholipase C1 C-terminal construct (PLC-ct) selectively reduced VI oxo-M modulation in PTX-treated and untreated cells. Expression of the G␥ buffers transducin (G␣ tr) and a G-protein-coupled-receptor kinase (GRK3) construct (MAS-GRK3) eliminated oxo-M modulation. Activation of the heterologously expressed neurokinin type 1 receptor, a G␣ q/11-coupled receptor, resulted in VI calcium current modulation. This modulation was eliminated with coexpression of G␣ tr or MAS-GRK3. Cells expressing G 1 ␥ 2 were tonically inhibited via the VD pathway. Application of oxo-M to these cells produced VI modulation and reduced the amount of current inhibited via the VD pathway. Together, these results confirm the requirement for G␥ in VD modulation and implicate G␣ q-GTP and G␥ as components in the potentially novel VI pathway.
Journal of Neurophysiology, 1997
Kammermeier, Paul J. and Stephen W. Jones. High-voltage-activated calcium currents in neurons acu... more Kammermeier, Paul J. and Stephen W. Jones. High-voltage-activated calcium currents in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus. J. Neurophysiol. 77: 465–475, 1997. We studied the high-voltage-activated (HVA) calcium currents in cells isolated from the ventrobasal nucleus of the rat thalamus with the use of the whole cell patch-clamp technique. Low-voltage-activated current was inactivated by the use of long voltage steps or 100-ms prepulses to −20 mV. We used channel blocking agents to characterize the currents that make up the HVA current. The dihydropyridine (DHP) antagonist nimodipine (5 μM) reversibly blocked 33 ± 1% (mean ± SE), and ω-conotoxin GVIA (1 μM) irreversibly blocked 25 ± 5%. The current resistant to DHPs and ω-conotoxin GVIA was inhibited almost completely by ω-conotoxin MVIIC (90 ± 5% at 3–5 μM) and was partially inhibited by ω-agatoxin IVA (54 ± 4% block at 1 μM). We conclude that there are at least four main HVA currents in thalami...
The Journal of Neuroscience, 2000
Group I metabotropic glutamate receptors (mGluR1 and 5) couple to intracellular calcium pools by ... more Group I metabotropic glutamate receptors (mGluR1 and 5) couple to intracellular calcium pools by a family of proteins, termed Homer, that cross-link the receptor to inositol trisphosphate receptors. mGluRs also couple to membrane ion channels via G-proteins. The role of Homer proteins in channel modulation was investigated by expressing mGluRs and various forms of Homer in rat superior cervical ganglion (SCG) sympathetic neurons by intranuclear cDNA injection. Expression of cross-linkingcapable forms of Homer (Homer 1b, 1c, 2, and 3, termed long forms) occluded group I mGluR-mediated N-type calcium and M-type potassium current modulation. This effect was specific for group I mGluRs. mGluR2 (group II)-mediated inhibition of N-channels was unaltered. Long forms of Homer decreased modulation of N-and M-type currents but did not selectively block distinct G-protein pathways. Short forms of Homer, which cannot self-multimerize (Homer 1a and a Homer 2 C-terminal deletion), did not alter mGluR-ion channel coupling. When coexpressed with long forms of Homer, short forms restored the mGluR1a-mediated calcium current modulation in an apparent dose-dependent manner. Homer 2b induced cell surface clusters of mGluR5 in SCG neurons. Conversely, a uniform distribution was observed when mGluR5 was expressed alone or with Homer short forms. These studies indicate that long and short forms of Homer compete for binding to mGluRs and regulate their coupling to ion channels. In vivo, the immediate early Homer 1a is anticipated to enhance ion channel modulation and to disrupt coupling to releasable intracellular calcium pools. Thus, Homer may regulate the magnitude and predominate signaling output of group I mGluRs.
Cold Spring Harbor protocols, Jan 5, 2017
From patch-clamp techniques to recombinant DNA technologies, three-dimensional protein modeling, ... more From patch-clamp techniques to recombinant DNA technologies, three-dimensional protein modeling, and optogenetics, diverse and sophisticated methods have been used to study ion channels and how they determine the electrical properties of cells.
Channels (Austin, Tex.), Jan 24, 2017
Type two voltage gated calcium (CaV2) channels are the primary mediators of neurotransmission at ... more Type two voltage gated calcium (CaV2) channels are the primary mediators of neurotransmission at neuronal presynapses, but their function at neural soma is also important in regulating excitability (1) . Mechanisms that regulate CaV2 channel expression at synapses have been studied extensively, which motivated us to perform similar studies in the soma. Rat sympathetic neurons from the superior cervical ganglion (SCG) natively express CaV2.2 and CaV2.3 (2) . We noted previously that heterologous expression of CaV2.1 but not CaV2.2 results in increased calcium current in SCG neurons (3) . In the present study, we extended these observations to show that both CaV2.1 and CaV2.3 expression resulted in increased calcium currents while CaV2.2 expression did not. Further, CaV2.1 could displace native CaV2.2 channels, but CaV2.3 expression could not. Heterologous expression of the individual accessory subunits α2δ-1, α2δ-2, α2δ-3, or β4 alone failed to increase current density, suggesting th...
The Journal of pharmacology and experimental therapeutics, 2017
In rat sympathetic neurons from the superior cervical ganglia (SCG) expressing metabotropic gluta... more In rat sympathetic neurons from the superior cervical ganglia (SCG) expressing metabotropic glutamate receptor mGluR1 or mGluR5, overexpression of scaffolding Homer proteins, which bind to a Homer ligand in their C termini, cause receptor clustering and uncoupling from ion channel modulation. In the absence of recombinant Homer protein overexpression, uncoupling of mGluRs from voltage-dependent channels can be induced by expression of Preso1, an adaptor of proline-directed kinases that phosphorylates the Homer ligand and recruits binding of endogenous Homer proteins. Here we show that in SCG neurons expressing mGluR1 and the tyrosine receptor kinase B, treatment with brain-derived neurotrophic factor (BDNF) produces a similar uncoupling of the receptors from calcium channels. We investigated the pathways that mediate this uncoupling and compared it with uncoupling observed with Preso1 expression. Both BDNF- and Preso1-induced uncoupling require residues T1151 and S1154 in the mGluR1...