Meera Pratap - Academia.edu (original) (raw)
Papers by Meera Pratap
Proceedings of the National Academy of Sciences, 2018
Significance The Src family of nonreceptor tyrosine kinases (SFK) is essential for nervous system... more Significance The Src family of nonreceptor tyrosine kinases (SFK) is essential for nervous system function and may contribute to neurodegeneration. Spinocerebellar ataxias (SCAs) are neurodegenerative diseases in which Purkinje neurons fire irregularly and degenerate leading to motor problems. We show that the SFK suppressor Missing-in-metastasis (MTSS1) is an ataxia gene that links multiple SCAs. MTSS1 loss results in increased SFK activity, degenerating Purkinje neurons with low firing rates, and cell death. Surprisingly, mouse models for three different SCAs show elevated SFK activity, with SCA1 and SCA2 models displaying dramatically reduced MTSS1 protein levels. Treatment of each SCA model with an SFK inhibitor corrects Purkinje basal firing and delays ataxia progression in MTSS1 mutants. Our results identify a common link among disparate neurodegenerative diseases.
Guanidinoacetate ( GAA ) Is a Potent GABA A Receptor GABA Mimetic: Implications for Neurological Disease Pathology
Journal of Neurochemistry
Physiology, 1998
Large-conductance, voltage-, and Ca2+-sensitive K+ (maxi-KCa) channels regulate neuronal and smoo... more Large-conductance, voltage-, and Ca2+-sensitive K+ (maxi-KCa) channels regulate neuronal and smooth muscle excitability. Their pore-forming α-subunit shows similarities with voltage-gated channels and indeed can open in the practical absence of Ca2+. The NH2 terminus is unique, with a seventh transmembrane segment involved in β-subunit modulation. The long COOH terminus is implied in Ca2+ modulation.
Wallner, M. et al. Characterization of and modulation by a -subunit of a human maxi KCa channel cloned from myometrium. Receptors Channels 3, 185−199
Receptors and Channels
ABSTRACT
Bravo-Zehnder, M. et al. Apical sorting of a voltage- and Ca2+-activated K+ channel - subunit in Madin-Darby canine kidney cells is independent of N-glycosylation. Proc. Natl Acad. Sci. USA 97, 13114-13119
Proceedings of the National Academy of Sciences
The voltage- and Ca(2+)-activated K(+) (K(V,Ca)) channel is expressed in a variety of polarized e... more The voltage- and Ca(2+)-activated K(+) (K(V,Ca)) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel K(V,Ca) alpha-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., K(V,Ca) beta-subunits) were not required. hSlo seems to display properties for li...
Chapter 8 Calcium-Activated Potassium Channels in Muscle and Brain
Potassium Ion Channels Molecular Structure, Function, and Diseases, 1999
ABSTRACT This chapter discusses the characterization of the two subfamilies that can account for ... more ABSTRACT This chapter discusses the characterization of the two subfamilies that can account for the variety of Ca2+-sensitive K+ channels: large conductance (BK) and small conductance (SK) channels. BK channels are characterized by their high single-channel conductance, their dual response to voltage and Ca2+, and their blockade by nanomolar concentrations of iberiotoxin and micromolar concentrations of TEA. Heterologously expressed α-subunit cDNA clones mirror these features. Structurally, they are characterized by unique N- and C-terminal sequences, implicated in β-subunit and Ca2+ regulation, appended to a six-transmembrane structure typical of voltage-gated ion channels. SK channels are characterized by their small conductance, their voltage independence, and their Ca2+-dependent activation. A family of this class of K+ channels has been recently cloned. BK and different types of SK channels are often coexpressed within the same native cell and seem to be coupled to specific Ca2+ sources. Specific Ca2+ channel blockers can abolish certain types of calcium-activated K channels without affecting others.
Protein Expression and Purification, 2006
The production of mammalian proteins in suYcient quantity and quality for structural and function... more The production of mammalian proteins in suYcient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a signiWcant number of mammalian proteins. This creates the necessity of well-identiWed expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the Xuorescent protein EGFP as a model, it is shown that muscle Wbers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of »1 mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.
Proceedings of the National Academy of Sciences, 1996
The pore-forming α subunit of large conductance voltage- and Ca 2+ -sensitive K (MaxiK) channels ... more The pore-forming α subunit of large conductance voltage- and Ca 2+ -sensitive K (MaxiK) channels is regulated by a β subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming α subunit necessary for β-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to β-subunit modulation, and analyzed the topology of the α subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel α subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1–S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation ...
Proceedings of the National Academy of Sciences, 2000
Large conductance voltage and Ca 2+ -activated K + (MaxiK) channels couple intracellular Ca 2+ wi... more Large conductance voltage and Ca 2+ -activated K + (MaxiK) channels couple intracellular Ca 2+ with cellular excitability. They are composed of a pore-forming α subunit and modulatory β subunits. The pore blockers charybdotoxin (CTx) and iberiotoxin (IbTx), at nanomolar concentrations, have been invaluable in unraveling MaxiK channel physiological role in vertebrates. However in mammalian brain, CTx-insensitive MaxiK channels have been described [Reinhart, P. H., Chung, S. & Levitan, I. B. (1989) Neuron 2, 1031–1041], but their molecular basis is unknown. Here we report a human MaxiK channel β-subunit (β4), highly expressed in brain, which renders the MaxiK channel α-subunit resistant to nanomolar concentrations of CTx and IbTx. The resistance of MaxiK channel to toxin block, a phenotype conferred by the β4 extracellular loop, results from a dramatic (≈1,000 fold) slowdown of the toxin association. However once bound, the toxin block is apparently irreversible. Thus, unusually high ...
Proceedings of the National Academy of Sciences, 1999
Voltage-dependent and calcium-sensitive K + (MaxiK) channels are key regulators of neuronal excit... more Voltage-dependent and calcium-sensitive K + (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca 2+ . In most tissues, their stimulation results in a noninactivating hyperpolarizing K + current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane β subunit (β2) that yields inactivating MaxiK currents on coexpression with the pore-forming α subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the β2 subunit abolished inactivation of the α subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle β1 subunit leads to an inactivating ph...
Proceedings of the National Academy of Sciences, 1997
Large conductance voltage- and Ca 2+ -dependent K + (MaxiK) channels show sequence similarities t... more Large conductance voltage- and Ca 2+ -dependent K + (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and “ in vivo ” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.
Molecular Pharmacology, 2010
GABA A receptors (GABA A Rs) have long been a focus as targets for alcohol actions. Recent work s... more GABA A receptors (GABA A Rs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic ␦ subunit-containing GABA A Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant ␣43␦ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of ␣43␦ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating ␦ subunits into functional receptors. To track ␦ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the ␦ subunit is replaced by an alanine residue found at the homologous position in ␥ subunits. We demonstrate that the ␦H68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive ␣43␦ receptors. The extent of enhancement of ␣43␦H68A receptors by 1 M diazepam, 30 mM EtOH, and 1 M -carboline-3-carboxy ethyl ester (but not 1 M Zn 2ϩ block) is correlated in individual recordings, suggesting that ␦ subunit incorporation into recombinant GABA A Rs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that ␦ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native ␦ subunitcontaining GABA A Rs.
Journal of Biological Chemistry, 1998
Native large conductance, voltage-dependent, and Ca 2؉-sensitive K ؉ channels are activated by cG... more Native large conductance, voltage-dependent, and Ca 2؉-sensitive K ؉ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human poreforming ␣-subunit of the Ca 2؉-sensitive K ؉ channel, Hslo, and the ␣-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by ϳ20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates ␥-33 P into the immunopurified Hslo band of ϳ125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the ␣-subunit of large conductance Ca 2؉-sensitive K ؉ channels is substrate for G-I␣ kinase in vivo and support direct phosphorylation as a mechanism for PKG-I␣-induced activation of maxi-K channels. Large conductance, voltage-dependent, and Ca 2ϩ-sensitive K ϩ (maxi-K) 1 channels are ubiquitously expressed, except in
FEBS Letters, 1993
Co-expression of cloned sodium channel p,-subunit with the rat skeletal muscle-subunit (Q) accele... more Co-expression of cloned sodium channel p,-subunit with the rat skeletal muscle-subunit (Q) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from cc&,, indicating that B, restores 'physiological' behaviour. Northern blot analysis revealed that the Na+ channel /$-subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the 'brain' B, cDNA to skeletal muscle and heart mRNA indicated that the diffferent Na' channel a-subunits in brain, skeletal muscle and heart may share a common /J-subunit.
FEBS Letters, 1996
Kv,c,[3 subunit dramatically increases the apparent calcium sensitivity of the eB subunlt of Maxi... more Kv,c,[3 subunit dramatically increases the apparent calcium sensitivity of the eB subunlt of MaxiK channels when probed in the mieremolar [Caa+h range, Analysis in a wide range of ICaa+h revealed that this functional coupling Is exquisitely modulated by [Caa+Jt. Ca a+ leas switch MaxIK a+iS complex Into a functionally coupled state at concentrations beyond resting lCaa+h. At IC_aZ+ls 100 .M, MaxlK activity becomes hidependent of Ca z÷, b purely voltqle.aetivated, and Its fuactleaai coupling with its_iS subunlt Is released. The funetiOllal switch develops at [CaZ+Jl that occur during cellular excitation, INre~ the moleeulm' bssis of how MaxiK channels regulate smooth muscle excitability and neuretransmitter release.
Human Gene Therapy, 2013
Spinal muscular atrophy (SMA) is an autosomal recessive disease affecting *1 in 10,000 live birth... more Spinal muscular atrophy (SMA) is an autosomal recessive disease affecting *1 in 10,000 live births. The most striking component is the loss of a-motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment paradigm other than supportive care, though the past 15 years has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease-modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials, including the application of antisense oligonucleotide (ASO) therapy for the correction of aberrant RNA splicing characteristic of SMA. Survival motor neuron (SMN) is a ubiquitously expressed 38-kD protein. Humans have two genes that produce SMN, SMN1 and SMN2, the former of which is deleted or nonfunctional in the majority of patients with SMA. These two genes are nearly identical with one exception, a C to T transition (C6T) within exon 7 of SMN2. C6T disrupts a modulator of splicing, leading to the exclusion of exon 7 from *90% of the mRNA transcript. The resultant truncated D7SMN protein does not oligomerize efficiently and is rapidly degraded. SMA can therefore be considered a disease of too little SMN protein. A number of cis-acting splice modifiers have been identified in the region of exon 7, the steric block of which enhances the retention of the exon and a resultant full-length mRNA sequence. ASOs targeted to these splice motifs have shown impressive phenotype rescue in multiple SMA mouse models.
Wallner, M. et al. Characterization of and modulation by a -subunit of a human maxi KCa channel cloned from myometrium. Receptors Channels 3, 185−199
Receptors and Channels
ABSTRACT
Proceedings of the National Academy of Sciences
The pore-forming ␣ subunit of large conductance voltage-and Ca 2؉ -sensitive K (MaxiK) channels i... more The pore-forming ␣ subunit of large conductance voltage-and Ca 2؉ -sensitive K (MaxiK) channels is regulated by a  subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming ␣ subunit necessary for -subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to -subunit modulation, and analyzed the topology of the ␣ subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel ␣ subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers -subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function.
The Journal of Physiology
1. Human large-conductance voltageand calcium-sensitive K+ (maxi KCa) channels are composed of at... more 1. Human large-conductance voltageand calcium-sensitive K+ (maxi KCa) channels are composed of at least two subunits: the pore-forming subunit, a, and a modulatory subunit, /1. Expression of the / subunit induces dramatic changes in a subunit function. It increases the apparent Ca2+ sensitivity and it allows dehydrosoyasaponin I (DHS-I) to upregulate the channel.
Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A
The American journal of physiology
Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activit... more Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activity. Ca(2+)-activated K+ channels (KCa) play a role in the modulation of uterine contractions and myometrial Ca2+ homeostasis and have been implicated in the control of smooth muscle excitability. We now show that relaxin stimulates KCa channels in cell-attached patches in a cell line derived from term pregnant human myometrium. This effect was prevented by the protein kinase A (PKA) antagonist, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). After patch excision, the channel was activated by PKA and inhibited by alkaline phosphatase. These data suggest that relaxin may promote myometrial quiescence in part by stimulation of KCa channels via a PKA-mediated mechanism.
Proceedings of the National Academy of Sciences, 2018
Significance The Src family of nonreceptor tyrosine kinases (SFK) is essential for nervous system... more Significance The Src family of nonreceptor tyrosine kinases (SFK) is essential for nervous system function and may contribute to neurodegeneration. Spinocerebellar ataxias (SCAs) are neurodegenerative diseases in which Purkinje neurons fire irregularly and degenerate leading to motor problems. We show that the SFK suppressor Missing-in-metastasis (MTSS1) is an ataxia gene that links multiple SCAs. MTSS1 loss results in increased SFK activity, degenerating Purkinje neurons with low firing rates, and cell death. Surprisingly, mouse models for three different SCAs show elevated SFK activity, with SCA1 and SCA2 models displaying dramatically reduced MTSS1 protein levels. Treatment of each SCA model with an SFK inhibitor corrects Purkinje basal firing and delays ataxia progression in MTSS1 mutants. Our results identify a common link among disparate neurodegenerative diseases.
Guanidinoacetate ( GAA ) Is a Potent GABA A Receptor GABA Mimetic: Implications for Neurological Disease Pathology
Journal of Neurochemistry
Physiology, 1998
Large-conductance, voltage-, and Ca2+-sensitive K+ (maxi-KCa) channels regulate neuronal and smoo... more Large-conductance, voltage-, and Ca2+-sensitive K+ (maxi-KCa) channels regulate neuronal and smooth muscle excitability. Their pore-forming α-subunit shows similarities with voltage-gated channels and indeed can open in the practical absence of Ca2+. The NH2 terminus is unique, with a seventh transmembrane segment involved in β-subunit modulation. The long COOH terminus is implied in Ca2+ modulation.
Wallner, M. et al. Characterization of and modulation by a -subunit of a human maxi KCa channel cloned from myometrium. Receptors Channels 3, 185−199
Receptors and Channels
ABSTRACT
Bravo-Zehnder, M. et al. Apical sorting of a voltage- and Ca2+-activated K+ channel - subunit in Madin-Darby canine kidney cells is independent of N-glycosylation. Proc. Natl Acad. Sci. USA 97, 13114-13119
Proceedings of the National Academy of Sciences
The voltage- and Ca(2+)-activated K(+) (K(V,Ca)) channel is expressed in a variety of polarized e... more The voltage- and Ca(2+)-activated K(+) (K(V,Ca)) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel K(V,Ca) alpha-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., K(V,Ca) beta-subunits) were not required. hSlo seems to display properties for li...
Chapter 8 Calcium-Activated Potassium Channels in Muscle and Brain
Potassium Ion Channels Molecular Structure, Function, and Diseases, 1999
ABSTRACT This chapter discusses the characterization of the two subfamilies that can account for ... more ABSTRACT This chapter discusses the characterization of the two subfamilies that can account for the variety of Ca2+-sensitive K+ channels: large conductance (BK) and small conductance (SK) channels. BK channels are characterized by their high single-channel conductance, their dual response to voltage and Ca2+, and their blockade by nanomolar concentrations of iberiotoxin and micromolar concentrations of TEA. Heterologously expressed α-subunit cDNA clones mirror these features. Structurally, they are characterized by unique N- and C-terminal sequences, implicated in β-subunit and Ca2+ regulation, appended to a six-transmembrane structure typical of voltage-gated ion channels. SK channels are characterized by their small conductance, their voltage independence, and their Ca2+-dependent activation. A family of this class of K+ channels has been recently cloned. BK and different types of SK channels are often coexpressed within the same native cell and seem to be coupled to specific Ca2+ sources. Specific Ca2+ channel blockers can abolish certain types of calcium-activated K channels without affecting others.
Protein Expression and Purification, 2006
The production of mammalian proteins in suYcient quantity and quality for structural and function... more The production of mammalian proteins in suYcient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a signiWcant number of mammalian proteins. This creates the necessity of well-identiWed expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the Xuorescent protein EGFP as a model, it is shown that muscle Wbers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of »1 mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.
Proceedings of the National Academy of Sciences, 1996
The pore-forming α subunit of large conductance voltage- and Ca 2+ -sensitive K (MaxiK) channels ... more The pore-forming α subunit of large conductance voltage- and Ca 2+ -sensitive K (MaxiK) channels is regulated by a β subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming α subunit necessary for β-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to β-subunit modulation, and analyzed the topology of the α subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel α subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1–S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation ...
Proceedings of the National Academy of Sciences, 2000
Large conductance voltage and Ca 2+ -activated K + (MaxiK) channels couple intracellular Ca 2+ wi... more Large conductance voltage and Ca 2+ -activated K + (MaxiK) channels couple intracellular Ca 2+ with cellular excitability. They are composed of a pore-forming α subunit and modulatory β subunits. The pore blockers charybdotoxin (CTx) and iberiotoxin (IbTx), at nanomolar concentrations, have been invaluable in unraveling MaxiK channel physiological role in vertebrates. However in mammalian brain, CTx-insensitive MaxiK channels have been described [Reinhart, P. H., Chung, S. & Levitan, I. B. (1989) Neuron 2, 1031–1041], but their molecular basis is unknown. Here we report a human MaxiK channel β-subunit (β4), highly expressed in brain, which renders the MaxiK channel α-subunit resistant to nanomolar concentrations of CTx and IbTx. The resistance of MaxiK channel to toxin block, a phenotype conferred by the β4 extracellular loop, results from a dramatic (≈1,000 fold) slowdown of the toxin association. However once bound, the toxin block is apparently irreversible. Thus, unusually high ...
Proceedings of the National Academy of Sciences, 1999
Voltage-dependent and calcium-sensitive K + (MaxiK) channels are key regulators of neuronal excit... more Voltage-dependent and calcium-sensitive K + (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca 2+ . In most tissues, their stimulation results in a noninactivating hyperpolarizing K + current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane β subunit (β2) that yields inactivating MaxiK currents on coexpression with the pore-forming α subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the β2 subunit abolished inactivation of the α subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle β1 subunit leads to an inactivating ph...
Proceedings of the National Academy of Sciences, 1997
Large conductance voltage- and Ca 2+ -dependent K + (MaxiK) channels show sequence similarities t... more Large conductance voltage- and Ca 2+ -dependent K + (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and “ in vivo ” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.
Molecular Pharmacology, 2010
GABA A receptors (GABA A Rs) have long been a focus as targets for alcohol actions. Recent work s... more GABA A receptors (GABA A Rs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic ␦ subunit-containing GABA A Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant ␣43␦ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of ␣43␦ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating ␦ subunits into functional receptors. To track ␦ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the ␦ subunit is replaced by an alanine residue found at the homologous position in ␥ subunits. We demonstrate that the ␦H68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive ␣43␦ receptors. The extent of enhancement of ␣43␦H68A receptors by 1 M diazepam, 30 mM EtOH, and 1 M -carboline-3-carboxy ethyl ester (but not 1 M Zn 2ϩ block) is correlated in individual recordings, suggesting that ␦ subunit incorporation into recombinant GABA A Rs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that ␦ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native ␦ subunitcontaining GABA A Rs.
Journal of Biological Chemistry, 1998
Native large conductance, voltage-dependent, and Ca 2؉-sensitive K ؉ channels are activated by cG... more Native large conductance, voltage-dependent, and Ca 2؉-sensitive K ؉ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human poreforming ␣-subunit of the Ca 2؉-sensitive K ؉ channel, Hslo, and the ␣-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by ϳ20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates ␥-33 P into the immunopurified Hslo band of ϳ125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the ␣-subunit of large conductance Ca 2؉-sensitive K ؉ channels is substrate for G-I␣ kinase in vivo and support direct phosphorylation as a mechanism for PKG-I␣-induced activation of maxi-K channels. Large conductance, voltage-dependent, and Ca 2ϩ-sensitive K ϩ (maxi-K) 1 channels are ubiquitously expressed, except in
FEBS Letters, 1993
Co-expression of cloned sodium channel p,-subunit with the rat skeletal muscle-subunit (Q) accele... more Co-expression of cloned sodium channel p,-subunit with the rat skeletal muscle-subunit (Q) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from cc&,, indicating that B, restores 'physiological' behaviour. Northern blot analysis revealed that the Na+ channel /$-subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the 'brain' B, cDNA to skeletal muscle and heart mRNA indicated that the diffferent Na' channel a-subunits in brain, skeletal muscle and heart may share a common /J-subunit.
FEBS Letters, 1996
Kv,c,[3 subunit dramatically increases the apparent calcium sensitivity of the eB subunlt of Maxi... more Kv,c,[3 subunit dramatically increases the apparent calcium sensitivity of the eB subunlt of MaxiK channels when probed in the mieremolar [Caa+h range, Analysis in a wide range of ICaa+h revealed that this functional coupling Is exquisitely modulated by [Caa+Jt. Ca a+ leas switch MaxIK a+iS complex Into a functionally coupled state at concentrations beyond resting lCaa+h. At IC_aZ+ls 100 .M, MaxlK activity becomes hidependent of Ca z÷, b purely voltqle.aetivated, and Its fuactleaai coupling with its_iS subunlt Is released. The funetiOllal switch develops at [CaZ+Jl that occur during cellular excitation, INre~ the moleeulm' bssis of how MaxiK channels regulate smooth muscle excitability and neuretransmitter release.
Human Gene Therapy, 2013
Spinal muscular atrophy (SMA) is an autosomal recessive disease affecting *1 in 10,000 live birth... more Spinal muscular atrophy (SMA) is an autosomal recessive disease affecting *1 in 10,000 live births. The most striking component is the loss of a-motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment paradigm other than supportive care, though the past 15 years has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease-modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials, including the application of antisense oligonucleotide (ASO) therapy for the correction of aberrant RNA splicing characteristic of SMA. Survival motor neuron (SMN) is a ubiquitously expressed 38-kD protein. Humans have two genes that produce SMN, SMN1 and SMN2, the former of which is deleted or nonfunctional in the majority of patients with SMA. These two genes are nearly identical with one exception, a C to T transition (C6T) within exon 7 of SMN2. C6T disrupts a modulator of splicing, leading to the exclusion of exon 7 from *90% of the mRNA transcript. The resultant truncated D7SMN protein does not oligomerize efficiently and is rapidly degraded. SMA can therefore be considered a disease of too little SMN protein. A number of cis-acting splice modifiers have been identified in the region of exon 7, the steric block of which enhances the retention of the exon and a resultant full-length mRNA sequence. ASOs targeted to these splice motifs have shown impressive phenotype rescue in multiple SMA mouse models.
Wallner, M. et al. Characterization of and modulation by a -subunit of a human maxi KCa channel cloned from myometrium. Receptors Channels 3, 185−199
Receptors and Channels
ABSTRACT
Proceedings of the National Academy of Sciences
The pore-forming ␣ subunit of large conductance voltage-and Ca 2؉ -sensitive K (MaxiK) channels i... more The pore-forming ␣ subunit of large conductance voltage-and Ca 2؉ -sensitive K (MaxiK) channels is regulated by a  subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming ␣ subunit necessary for -subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to -subunit modulation, and analyzed the topology of the ␣ subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel ␣ subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers -subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function.
The Journal of Physiology
1. Human large-conductance voltageand calcium-sensitive K+ (maxi KCa) channels are composed of at... more 1. Human large-conductance voltageand calcium-sensitive K+ (maxi KCa) channels are composed of at least two subunits: the pore-forming subunit, a, and a modulatory subunit, /1. Expression of the / subunit induces dramatic changes in a subunit function. It increases the apparent Ca2+ sensitivity and it allows dehydrosoyasaponin I (DHS-I) to upregulate the channel.
Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A
The American journal of physiology
Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activit... more Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activity. Ca(2+)-activated K+ channels (KCa) play a role in the modulation of uterine contractions and myometrial Ca2+ homeostasis and have been implicated in the control of smooth muscle excitability. We now show that relaxin stimulates KCa channels in cell-attached patches in a cell line derived from term pregnant human myometrium. This effect was prevented by the protein kinase A (PKA) antagonist, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). After patch excision, the channel was activated by PKA and inhibited by alkaline phosphatase. These data suggest that relaxin may promote myometrial quiescence in part by stimulation of KCa channels via a PKA-mediated mechanism.