Miguel Holmgren - Academia.edu (original) (raw)
Papers by Miguel Holmgren
PNAS Nexus
Na+/K+-ATPase, which creates transmembrane electrochemical gradients by exchanging 3 Na+ for 2 K+... more Na+/K+-ATPase, which creates transmembrane electrochemical gradients by exchanging 3 Na+ for 2 K+, is central to the pathogenesis of neurological diseases such as alternating hemiplegia of childhood. Although Na+/K+-ATPase has 3 distinct ion binding sites I–III, the difficulty of distinguishing ion binding events at each site from the others hinders kinetic study of these transitions. Here, we show that binding of Na+ at each site in the human α3 Na+/K+-ATPase can be resolved using extracellular Na+-mediated transient currents. When Na+/K+-ATPase is constrained to bind and release only Na+, three kinetic components: fast, medium, and slow, can be isolated, presumably corresponding to the protein dynamics associated with the binding (or release depending on the voltage step direction) and the occlusion (or deocclusion) of each of the 3 Na+. Patient-derived mutations of residues which coordinate Na+ at site III exclusively impact the slow component, demonstrating that site III is cruc...
Biophysical Journal, 2014
Journal of molecular biology, Jan 28, 2015
Using the energy of ATP hydrolysis, the Na(+)/K(+)-ATPase is able to transport across the cell me... more Using the energy of ATP hydrolysis, the Na(+)/K(+)-ATPase is able to transport across the cell membrane Na(+) and K(+) against their electrochemical gradients. The enzyme is strongly inhibited by ouabain and its derivatives, some that are therapeutically used for patients with heart failure (cardiotonic steroids). Using lanthanide resonance energy transfer (LRET), we trace here the conformational changes occurring on the external side of functional Na(+)/K(+)-ATPases induced by the binding of ouabain. Changes in donor/acceptor pair distances are mainly observed within the α subunit of the enzyme. To derive a structural model matching the experimental LRET distances measured with bound ouabain, molecular dynamic simulations were carried out with energy restraints applied simultaneously using a novel methodology with multiple non-interacting fragments. The restrained simulation, initiated from the X-ray structure of the E2(2K(+)) state, became strikingly similar to the X-ray structure...
Annals of the New York Academy of Sciences, 1997
Nature Communications, 2014
Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electr... more Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that statedependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open-and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.
Biophysical Journal, 2012
Biophysical Journal, 2013
molecular basis for how these chromatin enzymes recognize the 200 kDa nucleosome is largely unkno... more molecular basis for how these chromatin enzymes recognize the 200 kDa nucleosome is largely unknown. My laboratory investigates how chromatin enzymes interact with the nucleosome. We determined the first crystal structure of a chromatin protein in complex with the nucleosome. Our structure of the 300 kDa RCC1/nucleosome core particle complex at 2.9 Å resolution explains how RCC1's b-propeller domain recognizes the architecture of the nucleosome through a combination of both protein-protein and protein-DNA interactions. Our crystal structure also provides a first atomic view of the nucleosome core particle containing the Widom 601 nucleosome positioning sequence. We find that the 601 DNA forms a 145 bp nucleosome core particle and is thus overwound compared to the human alpha-satellite DNA used in prior nucleosome crystals. I will also discuss the molecular basis for why the Widom 601 DNA sequence is a strong nucleosome positioning sequence.
Biophysical Journal, 2020
The Na þ /K þ-ATPase is a chemical molecular machine responsible for the movement of Na þ and K þ... more The Na þ /K þ-ATPase is a chemical molecular machine responsible for the movement of Na þ and K þ ions across the cell membrane. These ions are moved against their electrochemical gradients, so the protein uses the free energy of ATP hydrolysis to transport them. In fact, the Na þ /K þ-ATPase is the single largest consumer of energy in most cells. In each pump cycle, the protein sequentially exports 3Na þ out of the cell, then imports 2K þ into the cell at an approximate rate of 200 cycles/s. In each half cycle of the transport process, there is a state in which ions are stably trapped within the permeation pathway of the protein by internal and external gates in their closed states. These gates are required to open alternately; otherwise, passive ion diffusion would be a wasteful end of the cell's energy. Once one of these gates open, ions diffuse from their binding sites to the accessible milieu, which involves moving through part of the electrical field across the membrane. Consequently, ions generate transient electrical currents first discovered more than 30 years ago. They have been studied in a variety of preparations, including native and heterologous expression systems. Here, we review three decades' worth of work using these transient electrical signals to understand the kinetic transitions of the movement of Na þ and K þ ions through the Na þ /K þ-ATPase and propose the significance that this work might have to the understanding of the dysfunction of human pump orthologs responsible for some newly discovered neurological pathologies.
Nature Structural & Molecular Biology, 2004
Potassium channels of the K V 1 subfamily play an important role in excitability by repolarizing ... more Potassium channels of the K V 1 subfamily play an important role in excitability by repolarizing membranes and shaping the firing properties of neurons 1. They are subject to fine-tuning through a number of regulatory mechanisms, including RNA editing. In fact, editing of K + channels is important enough to have been conserved over considerable phylogenetic distances. Representatives from diverse taxa such as squid 2,3 , flies 4 , mice 4 and human 4 all edit their K + channels. The reasons why the channels are modified, however, remain poorly understood because little is known about how the modifications affect channel function. Even less is known about how the process is regulated. Genomic recoding by RNA editing is catalyzed by a family of adenosine deaminases acting on RNA (ADARs) that specifically recognize adenosines within double-stranded regions of precursor mRNAs, converting them to inosine, a nucleotide that is interpreted as guanosine by the cellular machinery, including the ribosome 5. In all known cases, the double-stranded RNA structures are formed by base-pairing between residues in the vicinity of the editing site with residues in a neighboring intron 6-10. It is therefore notable to find that hK V 1.1, an intronless gene, is edited 4. We report here that a fully exonic hairpin structure directs efficient editing of hK V 1.1 mRNA by human adenosine deaminase acting on RNA 2 (hADAR2). Voltage-dependent K + channels modulate excitability by opening and closing a K +-selective pore in response to voltage. In many cases, K + ion flow can be interrupted when an intracellular particle occludes the pore, a process known as fast inactivation. In hK V 1.1 mRNAs, ADAR modification causes an isoleucine/valine (I/V) recoding event at a single position, Ile400, effectively converting the genomic isoleucine codon (ATT) to that of valine (GTT). The affected amino
Journal of Comparative Neurology, 2021
Mutations in genes encoding Na+ /K+ -ATPase α1, α2, and α3 subunits cause a wide range of disabli... more Mutations in genes encoding Na+ /K+ -ATPase α1, α2, and α3 subunits cause a wide range of disabling neurological disorders, and dysfunction of Na+ /K+ -ATPase may contribute to neuronal injury in stroke and dementia. To better understand the pathogenesis of these diseases, it is important to determine the expression patterns of the different Na+ /K+ -ATPase subunits within the brain and among specific cell types. Using two available scRNA-Seq databases from the adult mouse nervous system, we examined the mRNA expression patterns of the different isoforms of the Na+ /K+ -ATPase α, β and Fxyd subunits at the single cell level among brain regions and various neuronal populations. We subsequently identified specific types of neurons enriched with transcripts for α1 and α3 isoforms and elaborated how α3-expressing neuronal populations govern cerebellar neuronal circuits. We further analyzed the co-expression network for α1 and α3 isoforms, highlighting the genes that positively correlated with α1 and α3 expression. The top ten genes for α1 were Chn2, Hpcal1, Nrgn, Neurod1, Selm, Kcnc1, Snrk, Snap25, Ckb and Ccndbp1 and for α3 were Sorcs3, Eml5, Neurod2, Ckb, Tbc1d4, Ptprz1, Pvrl1, Kirrel3, Pvalb, and Asic2.
Neuron, 1998
In our previous investigation of the activation gating mechanism of voltage-activated K ϩ channel... more In our previous investigation of the activation gating mechanism of voltage-activated K ϩ channels, we studied the state-dependent accessibility of cysteines substituted at specific positions in the S6 region (Liu et al., 1997). Several S6 cysteines (470, 474, 476, and 478
Biophysical Journal, 2014
Biophysical Journal, 2014
Nature Communications, 2012
na + /K + pumps move net charge through the cell membrane by mediating unequal exchange of intrac... more na + /K + pumps move net charge through the cell membrane by mediating unequal exchange of intracellular na + and extracellular K +. most charge moves during transitions that release na + to the cell exterior. When pumps are constrained to bind and release only na + , a membrane voltage-step redistributes pumps among conformations with zero, one, two or three bound na + , thereby transiently generating current. By applying rapid voltage steps to squid giant axons, we previously identified three components in such transient currents, with distinct relaxation speeds: fast (which nearly parallels the voltage-jump time course), medium speed (τ m = 0.2-0.5 ms) and slow (τ s = 1-10 ms). Here we show that these three components are tightly correlated, both in their magnitudes and in the time courses of their changes. The correlations reveal the dynamics of the conformational rearrangements that release three na + to the exterior (or sequester them into their binding sites) one at a time, in an obligatorily sequential manner.
Biophysical Journal, 2015
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2008
By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleo... more By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleotide-gated (CNG) channels serve key roles in the transduction of visual and olfactory signals. Compiling evidence suggests that the activation gate in CNG channels is not located at the intracellular end of pore, as it has been established for voltage-activated potassium (K(V)) channels. Here, we show that ion permeation in CNG channels is tightly regulated at the selectivity filter. By scanning the entire selectivity filter using small cysteine reagents, like cadmium and silver, we observed a state-dependent accessibility pattern consistent with gated access at the middle of the selectivity filter, likely at the corresponding position known to regulate structural changes in KcsA channels in response to low concentrations of permeant ions.
Proceedings of the National Academy of Sciences, 2013
Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important ... more Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a “gating-ring” structure has been proposed to transduce Ca 2+ binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca 2+ binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.
Proceedings of the National Academy of Sciences, 2011
The Na + /K + pump is a nearly ubiquitous membrane protein in animal cells that uses the free ene... more The Na + /K + pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na + from the cell and import 2K + per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na + /K + pump that represent the kinetics of extracellular Na + binding/release and Na + occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas , we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associat...
PNAS Nexus
Na+/K+-ATPase, which creates transmembrane electrochemical gradients by exchanging 3 Na+ for 2 K+... more Na+/K+-ATPase, which creates transmembrane electrochemical gradients by exchanging 3 Na+ for 2 K+, is central to the pathogenesis of neurological diseases such as alternating hemiplegia of childhood. Although Na+/K+-ATPase has 3 distinct ion binding sites I–III, the difficulty of distinguishing ion binding events at each site from the others hinders kinetic study of these transitions. Here, we show that binding of Na+ at each site in the human α3 Na+/K+-ATPase can be resolved using extracellular Na+-mediated transient currents. When Na+/K+-ATPase is constrained to bind and release only Na+, three kinetic components: fast, medium, and slow, can be isolated, presumably corresponding to the protein dynamics associated with the binding (or release depending on the voltage step direction) and the occlusion (or deocclusion) of each of the 3 Na+. Patient-derived mutations of residues which coordinate Na+ at site III exclusively impact the slow component, demonstrating that site III is cruc...
Biophysical Journal, 2014
Journal of molecular biology, Jan 28, 2015
Using the energy of ATP hydrolysis, the Na(+)/K(+)-ATPase is able to transport across the cell me... more Using the energy of ATP hydrolysis, the Na(+)/K(+)-ATPase is able to transport across the cell membrane Na(+) and K(+) against their electrochemical gradients. The enzyme is strongly inhibited by ouabain and its derivatives, some that are therapeutically used for patients with heart failure (cardiotonic steroids). Using lanthanide resonance energy transfer (LRET), we trace here the conformational changes occurring on the external side of functional Na(+)/K(+)-ATPases induced by the binding of ouabain. Changes in donor/acceptor pair distances are mainly observed within the α subunit of the enzyme. To derive a structural model matching the experimental LRET distances measured with bound ouabain, molecular dynamic simulations were carried out with energy restraints applied simultaneously using a novel methodology with multiple non-interacting fragments. The restrained simulation, initiated from the X-ray structure of the E2(2K(+)) state, became strikingly similar to the X-ray structure...
Annals of the New York Academy of Sciences, 1997
Nature Communications, 2014
Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electr... more Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that statedependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open-and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.
Biophysical Journal, 2012
Biophysical Journal, 2013
molecular basis for how these chromatin enzymes recognize the 200 kDa nucleosome is largely unkno... more molecular basis for how these chromatin enzymes recognize the 200 kDa nucleosome is largely unknown. My laboratory investigates how chromatin enzymes interact with the nucleosome. We determined the first crystal structure of a chromatin protein in complex with the nucleosome. Our structure of the 300 kDa RCC1/nucleosome core particle complex at 2.9 Å resolution explains how RCC1's b-propeller domain recognizes the architecture of the nucleosome through a combination of both protein-protein and protein-DNA interactions. Our crystal structure also provides a first atomic view of the nucleosome core particle containing the Widom 601 nucleosome positioning sequence. We find that the 601 DNA forms a 145 bp nucleosome core particle and is thus overwound compared to the human alpha-satellite DNA used in prior nucleosome crystals. I will also discuss the molecular basis for why the Widom 601 DNA sequence is a strong nucleosome positioning sequence.
Biophysical Journal, 2020
The Na þ /K þ-ATPase is a chemical molecular machine responsible for the movement of Na þ and K þ... more The Na þ /K þ-ATPase is a chemical molecular machine responsible for the movement of Na þ and K þ ions across the cell membrane. These ions are moved against their electrochemical gradients, so the protein uses the free energy of ATP hydrolysis to transport them. In fact, the Na þ /K þ-ATPase is the single largest consumer of energy in most cells. In each pump cycle, the protein sequentially exports 3Na þ out of the cell, then imports 2K þ into the cell at an approximate rate of 200 cycles/s. In each half cycle of the transport process, there is a state in which ions are stably trapped within the permeation pathway of the protein by internal and external gates in their closed states. These gates are required to open alternately; otherwise, passive ion diffusion would be a wasteful end of the cell's energy. Once one of these gates open, ions diffuse from their binding sites to the accessible milieu, which involves moving through part of the electrical field across the membrane. Consequently, ions generate transient electrical currents first discovered more than 30 years ago. They have been studied in a variety of preparations, including native and heterologous expression systems. Here, we review three decades' worth of work using these transient electrical signals to understand the kinetic transitions of the movement of Na þ and K þ ions through the Na þ /K þ-ATPase and propose the significance that this work might have to the understanding of the dysfunction of human pump orthologs responsible for some newly discovered neurological pathologies.
Nature Structural & Molecular Biology, 2004
Potassium channels of the K V 1 subfamily play an important role in excitability by repolarizing ... more Potassium channels of the K V 1 subfamily play an important role in excitability by repolarizing membranes and shaping the firing properties of neurons 1. They are subject to fine-tuning through a number of regulatory mechanisms, including RNA editing. In fact, editing of K + channels is important enough to have been conserved over considerable phylogenetic distances. Representatives from diverse taxa such as squid 2,3 , flies 4 , mice 4 and human 4 all edit their K + channels. The reasons why the channels are modified, however, remain poorly understood because little is known about how the modifications affect channel function. Even less is known about how the process is regulated. Genomic recoding by RNA editing is catalyzed by a family of adenosine deaminases acting on RNA (ADARs) that specifically recognize adenosines within double-stranded regions of precursor mRNAs, converting them to inosine, a nucleotide that is interpreted as guanosine by the cellular machinery, including the ribosome 5. In all known cases, the double-stranded RNA structures are formed by base-pairing between residues in the vicinity of the editing site with residues in a neighboring intron 6-10. It is therefore notable to find that hK V 1.1, an intronless gene, is edited 4. We report here that a fully exonic hairpin structure directs efficient editing of hK V 1.1 mRNA by human adenosine deaminase acting on RNA 2 (hADAR2). Voltage-dependent K + channels modulate excitability by opening and closing a K +-selective pore in response to voltage. In many cases, K + ion flow can be interrupted when an intracellular particle occludes the pore, a process known as fast inactivation. In hK V 1.1 mRNAs, ADAR modification causes an isoleucine/valine (I/V) recoding event at a single position, Ile400, effectively converting the genomic isoleucine codon (ATT) to that of valine (GTT). The affected amino
Journal of Comparative Neurology, 2021
Mutations in genes encoding Na+ /K+ -ATPase α1, α2, and α3 subunits cause a wide range of disabli... more Mutations in genes encoding Na+ /K+ -ATPase α1, α2, and α3 subunits cause a wide range of disabling neurological disorders, and dysfunction of Na+ /K+ -ATPase may contribute to neuronal injury in stroke and dementia. To better understand the pathogenesis of these diseases, it is important to determine the expression patterns of the different Na+ /K+ -ATPase subunits within the brain and among specific cell types. Using two available scRNA-Seq databases from the adult mouse nervous system, we examined the mRNA expression patterns of the different isoforms of the Na+ /K+ -ATPase α, β and Fxyd subunits at the single cell level among brain regions and various neuronal populations. We subsequently identified specific types of neurons enriched with transcripts for α1 and α3 isoforms and elaborated how α3-expressing neuronal populations govern cerebellar neuronal circuits. We further analyzed the co-expression network for α1 and α3 isoforms, highlighting the genes that positively correlated with α1 and α3 expression. The top ten genes for α1 were Chn2, Hpcal1, Nrgn, Neurod1, Selm, Kcnc1, Snrk, Snap25, Ckb and Ccndbp1 and for α3 were Sorcs3, Eml5, Neurod2, Ckb, Tbc1d4, Ptprz1, Pvrl1, Kirrel3, Pvalb, and Asic2.
Neuron, 1998
In our previous investigation of the activation gating mechanism of voltage-activated K ϩ channel... more In our previous investigation of the activation gating mechanism of voltage-activated K ϩ channels, we studied the state-dependent accessibility of cysteines substituted at specific positions in the S6 region (Liu et al., 1997). Several S6 cysteines (470, 474, 476, and 478
Biophysical Journal, 2014
Biophysical Journal, 2014
Nature Communications, 2012
na + /K + pumps move net charge through the cell membrane by mediating unequal exchange of intrac... more na + /K + pumps move net charge through the cell membrane by mediating unequal exchange of intracellular na + and extracellular K +. most charge moves during transitions that release na + to the cell exterior. When pumps are constrained to bind and release only na + , a membrane voltage-step redistributes pumps among conformations with zero, one, two or three bound na + , thereby transiently generating current. By applying rapid voltage steps to squid giant axons, we previously identified three components in such transient currents, with distinct relaxation speeds: fast (which nearly parallels the voltage-jump time course), medium speed (τ m = 0.2-0.5 ms) and slow (τ s = 1-10 ms). Here we show that these three components are tightly correlated, both in their magnitudes and in the time courses of their changes. The correlations reveal the dynamics of the conformational rearrangements that release three na + to the exterior (or sequester them into their binding sites) one at a time, in an obligatorily sequential manner.
Biophysical Journal, 2015
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2008
By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleo... more By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleotide-gated (CNG) channels serve key roles in the transduction of visual and olfactory signals. Compiling evidence suggests that the activation gate in CNG channels is not located at the intracellular end of pore, as it has been established for voltage-activated potassium (K(V)) channels. Here, we show that ion permeation in CNG channels is tightly regulated at the selectivity filter. By scanning the entire selectivity filter using small cysteine reagents, like cadmium and silver, we observed a state-dependent accessibility pattern consistent with gated access at the middle of the selectivity filter, likely at the corresponding position known to regulate structural changes in KcsA channels in response to low concentrations of permeant ions.
Proceedings of the National Academy of Sciences, 2013
Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important ... more Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a “gating-ring” structure has been proposed to transduce Ca 2+ binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca 2+ binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.
Proceedings of the National Academy of Sciences, 2011
The Na + /K + pump is a nearly ubiquitous membrane protein in animal cells that uses the free ene... more The Na + /K + pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na + from the cell and import 2K + per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na + /K + pump that represent the kinetics of extracellular Na + binding/release and Na + occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas , we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associat...