Damian C Bell | University College London (original) (raw)

Papers by Damian C Bell

Research paper thumbnail of 19th World Congress of Basic and Clinical Pharmacology, July 2nd–7th, 2023, Glasgow

Bioelectricity, Aug 31, 2023

Research paper thumbnail of Use of automated patch clamp in cardiac safety assessment: past, present and future perspectives

Journal of Pharmacological and Toxicological Methods, Jul 1, 2021

There is no doubt that automated patch clamp (APC) technology has revolutionized research in biom... more There is no doubt that automated patch clamp (APC) technology has revolutionized research in biomedical science. High throughput ion channel screening is now an integral part of the development and safety profiling of the majority of new chemical entities currently developed to address unmet medical needs. The increased throughput it provides has significantly improved the ability to overcome the time-consuming, low throughput bottlenecks resulting from the more conventional manual patch clamp method, considered the 'gold standard', for studying ion channel function and pharmacology. While systems offering the luxury of automation have only been commercially available for two decades, the road leading to this new technology is long and rich in seminal, hands-on, studies dating back as far as the 18th century. So where does this technology currently stand, and what will it look like in the future? In the current article, we review the scientific history leading to the development of APC systems, examine key drivers in the rapid development of this technology (such as failed ion channel programmes and the issue of drug-induced hERG inhibition and QT interval prolongation), highlight key capabilities and finally provide some perspective on the current and future impact of the technology on cardiac safety assessment and biomedical science.

Research paper thumbnail of Advances in ion channel high throughput screening: where are we in 2023?

Expert Opinion on Drug Discovery, Dec 17, 2023

Research paper thumbnail of News and views on ion channels in cancer: is cancer a channelopathy?

Frontiers in Pharmacology, Nov 13, 2023

Research paper thumbnail of Adventures and Advances in Time Travel With Induced Pluripotent Stem Cells and Automated Patch Clamp

Frontiers in Molecular Neuroscience

In the Hollywood blockbuster “The Curious Case of Benjamin Button” a fantastical fable unfolds of... more In the Hollywood blockbuster “The Curious Case of Benjamin Button” a fantastical fable unfolds of a man’s life that travels through time reversing the aging process; as the tale progresses, the frail old man becomes a vigorous, vivacious young man, then man becomes boy and boy becomes baby. The reality of cellular time travel, however, is far more wondrous: we now have the ability to both reverse and then forward time on mature cells. Four proteins were found to rewind the molecular clock of adult cells back to their embryonic, “blank canvas” pluripotent stem cell state, allowing these pluripotent stem cells to then be differentiated to fast forward their molecular clocks to the desired adult specialist cell types. These four proteins – the “Yamanaka factors” – form critical elements of this cellular time travel, which deservedly won Shinya Yamanaka the Nobel Prize for his lab’s work discovering them. Human induced pluripotent stem cells (hiPSCs) hold much promise in our understandi...

Research paper thumbnail of Novel oral voltage-gated sodium channel (Na v ) inhibitors inhibit capsaicin induced cough in conscious guinea pigs

European Respiratory Journal, 2015

Rationale: Intractable chronic cough is a serious unmet clinical problem and new therapies are ne... more Rationale: Intractable chronic cough is a serious unmet clinical problem and new therapies are needed for this condition. Na v channels are key for nerve conduction. Local anesthetics that are known to block all Na v channels are effective antitussive agents but their non-selectivity precludes their regular use in patients due to their side effects. Aim and Objectives: Our aim was to compare two novel and selective oral Na v inhibitors vs. a non-selective reference Na v inhibitor in an animal cough model. Methods: Compound potency in Na v channels was assessed using a IonWorks Quattro Automated Patch Clamp System (IC50). Cough was induced in conscious guinea pigs by exposure to aerosolized capsaicin (30µM) and cough numbers were recorded. Results: Compound A showed a dual Na v 1.7-1.8 selectivity with IC50 of 0.5 and 2.6µM, respectively, while compound B was a Na v 1.7 selective compound (IC50 0.07µM). Both compounds inhibited the capsaicin induced cough at similar levels than the pan Na v inhibitor GSK 2338345 (compound A 70%; Compound B 60%; GSK 2338345 71% at 30mg/kg p.o.). Conclusions: These two novel selective oral Na v inhibitors from two distinct chemical series are able to significantly reduce capsaicin induced cough in guinea pigs at similar levels than the non-selective reference compound GSK 2338345. Selective oral Na v inhibitors may be a valuable therapy for chronic cough with lower potential for adverse effects.

Research paper thumbnail of On the perspective of an aging population and its potential impact on drug attrition and pre-clinical cardiovascular safety assessment

Journal of Pharmacological and Toxicological Methods

Research paper thumbnail of Electrophysiological investigations into the G-protein modulation of the neuronal L-type (alpha1D, Cav1.3) and N-type (alpha1B Cav2.2) voltage dependent calcium channels

Doctoral thesis, UCL (University College London)., 2000

Research paper thumbnail of Changes in Local S4 Environment Provide a Voltage-sensing Mechanism for Mammalian Hyperpolarization–activated HCN Channels

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function ... more The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH 2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitution...

Research paper thumbnail of Advancing Ion Channel Research with Automated Patch Clamp (APC) Electrophysiology Platforms

Ion Channels in Biophysics and Physiology, 2021

Since its development on the cusp of the new millennium, automated patch clamp (APC) technology h... more Since its development on the cusp of the new millennium, automated patch clamp (APC) technology has matured over the last two decades. The increased throughput it afforded promised a new paradigm in ion channel recordings: It offered the potential to overcome the time-consuming, low-throughput bottleneck arising from manual patch clamp (MPC) investigations. This chapter highlights the advances in technology, showing how APC platforms have 'democratised' ion channel recordings, lowering the technical bar whilst substantially raising throughput. It will describe the background of the seminal first-generation and updates on advances in second-generation platforms. Furthermore, the chapter summarises the advances APC has made in ion channel studies, including finding new tool compounds and medicines. New functionality and applications on APC platforms give ion channel researchers flexible tools to study ion channels with high quality and high throughput.

Research paper thumbnail of Calcium Channel b Subunit Promotes Voltage-Dependent Modulation of a 1 B by G bg

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming a1 subu... more Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming a1 subunit and auxiliary subunits, including the intracellular b subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free Gbg dimer concentration. Here we have examined the interaction between the regulation of N-type (a1B) channels by their b subunits and by Gbg dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC b subunit, we found a significantly larger Gbg-dependent inhibition of a1B channel activation when the VDCC a1B and b subunits were coexpressed. In the absence of coexpressed VDCC b subunit, the Gbg dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channe...

Research paper thumbnail of Use of automated patch clamp in cardiac safety assessment: Past, present future perspectives

Journal of Pharmacological and Toxicological Methods

Research paper thumbnail of Pain-Related Ion Channel Research : Insights from Industry and Academia

Automated patch clamp (APC) technology was first developed at the turn of the millennium. The inc... more Automated patch clamp (APC) technology was first developed at the turn of the millennium. The increased throughput it afforded promised a new paradigm in ion channel recordings: it offered the potential to overcome the time-consuming, lowthroughput bottleneck arising from manual patch clamp (MPC) investigations. This has relevance to the fast-paced development of novel therapies for chronic pain. This review highlights the advances in technology, using select examples, that have facilitated APC usage in both industry and academia. It covers both first generation and the latest developments in second-generation platforms. In addition, it also provides an overview of the pain research field and how APC platforms have furthered our understanding of ion channel research and the development of pharmacological tools and therapeutics. APC platforms have much to offer the ion channel research community and this review highlights areas of ‘best practice’ for both academia and industry. The i...

Research paper thumbnail of Generating Potent and Selective Inhibitors of Kv1.3 Ion Channel by Fusing Knottins (Venom Derived Mini Proteins) into Peripheral CDR Loops of Antibodies

Biophysical Journal

surface to a large cytoplasmic cavity, and a unique configuration of the photoactive site likely ... more surface to a large cytoplasmic cavity, and a unique configuration of the photoactive site likely responsible for the delayed Schiff base deprotonation. Using this structural information, we have identified residues that determine absorption wavelength and current-voltage dependence. Our results provide clues for rational engineering of ACR molecules further to increase their optogenetic utility.

Research paper thumbnail of Knotbodies: A New Generation of Ion Channel Therapeutic Biologics Created by Fusing Knottin Toxins into Antibodies

Biophysical Journal

and perinexal regions. We correlated nano-structural organisation of junctions to their function,... more and perinexal regions. We correlated nano-structural organisation of junctions to their function, as niches for Na v 1.5. Scanning ion conductance microscopy resolved junctional topography in neonatal rat ventricular cardiomyocyte. This was combined with cellattached Na v 1.5 recordings from cardiomyocyte-cardiomyocyte and cardiomyocyte-fibroblasts junctions. Functional and dominant-negative Cx43 EGFP adenoviruses were used to probe active/inactive junctions. The Na v b1 adhesion inhibitor peptide (badp1) was tested. Whole-cell Na v -currents were recorded to separate non-junctional badp1 effects. To assess transdepolarisation, current-clamp and optical measurements were combined. Plasma membranes were analysed for junctional protein density. Exogenously-delivered Cx43 EGFP trafficked to regions where Na v activity and cluster size were increased relative to native Cx43-junctions (p=0.015). Smaller Na v -clusters (5-10) were abundant at native junctions, larger clusters (20-30) predominated at Cx43 EGFP -junctions. Acute badp1 peptide treatment caused Na v downregulation at native (p<0.01) and Cx43 EGFP -rich junctions

Research paper thumbnail of Using automated patch clamp electrophysiology platforms in pain-related ion channel research: insights from industry and academia

British Journal of Pharmacology

Automated patch clamp (APC) technology was first developed at the turn of the millennium. The inc... more Automated patch clamp (APC) technology was first developed at the turn of the millennium. The increased throughput it afforded promised a new paradigm in ion channel recordings: it offered the potential to overcome the time-consuming, lowthroughput bottleneck arising from manual patch clamp (MPC) investigations. This has relevance to the fast-paced development of novel therapies for chronic pain. This review highlights the advances in technology, using select examples, that have facilitated APC usage in both industry and academia. It covers both first generation and the latest developments in second-generation platforms. In addition, it also provides an overview of the pain research field and how APC platforms have furthered our understanding of ion channel research and the development of pharmacological tools and therapeutics. APC platforms have much to offer the ion channel research community and this review highlights areas of 'best practice' for both academia and industry. The impact of APC platforms and the prospects for chronic pain ion channel research and improved therapeutics will be evaluated.

Research paper thumbnail of Probing S4 and S5 segment proximity in mammalian hyperpolarization-activated HCN channels by disulfide bridging and Cd2+ coordination

Pflügers Archiv - European Journal of Physiology, 2009

We explored the structural basis of voltage sensing in HCN1 hyperpolarization-activated channels ... more We explored the structural basis of voltage sensing in HCN1 hyperpolarization-activated channels by examining the relative orientation of the voltage-sensor and pore domains. The opening of channels engineered to contain single cysteine residues at the extracellular ends of the voltagesensing S4 (V246C) and pore-forming S5 (C303) domains is inhibited by formation of disulfide or cysteine:Cd 2+ bonds. As Cd 2+ coordination is promoted by depolarization, the S4-S5 interaction occurs preferentially in the closed state. The failure of oxidation to catalyze dimer formation, as assayed by Western blotting, indicates the V246C:C303 interaction occurs within a subunit. Intriguingly, a similar interaction has been observed in depolarization-activated Shaker Kv channels at depolarized potentials but such an intrasubunit interaction is inconsistent with the X-ray crystal structure of Kv1.2, wherein S4 approaches S5 of an adjacent subunit. These findings suggest channels of opposite voltage-sensing polarity adopt a conserved S4-S5 orientation in the depolarized state that is distinct from that trapped upon crystallization.

Research paper thumbnail of ω-conotoxin MVIIC reversibly inhibits a human N-type calcium channel and calcium influx into chick synaptosomes

Neuropharmacology, 1994

ABSTRACT

Research paper thumbnail of Changes in Local S4 Environment Provide a Voltage-sensing Mechanism for Mammalian Hyperpolarization-activated HCN Channels

The Journal of General Physiology, 2003

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function ... more The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions...

Research paper thumbnail of Calcium Channel β Subunit Promotes Voltage-Dependent Modulation of α1B by Gβγ

Biophysical Journal, 2000

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming ␣1 subu... more Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming ␣1 subunit and auxiliary subunits, including the intracellular ␤ subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G␤␥ dimer concentration. Here we have examined the interaction between the regulation of N-type (␣1B) channels by their ␤ subunits and by G␤␥ dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC ␤ subunit, we found a significantly larger G␤␥-dependent inhibition of ␣1B channel activation when the VDCC ␣1B and ␤ subunits were coexpressed. In the absence of coexpressed VDCC ␤ subunit, the G␤␥ dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC ␤ subunit coexpression restored all of the hallmarks of G␤␥ modulation. These results suggest that the VDCC ␤ subunit must be present for G␤␥ to induce voltage-dependent modulation of N-type calcium channels.

Research paper thumbnail of 19th World Congress of Basic and Clinical Pharmacology, July 2nd–7th, 2023, Glasgow

Bioelectricity, Aug 31, 2023

Research paper thumbnail of Use of automated patch clamp in cardiac safety assessment: past, present and future perspectives

Journal of Pharmacological and Toxicological Methods, Jul 1, 2021

There is no doubt that automated patch clamp (APC) technology has revolutionized research in biom... more There is no doubt that automated patch clamp (APC) technology has revolutionized research in biomedical science. High throughput ion channel screening is now an integral part of the development and safety profiling of the majority of new chemical entities currently developed to address unmet medical needs. The increased throughput it provides has significantly improved the ability to overcome the time-consuming, low throughput bottlenecks resulting from the more conventional manual patch clamp method, considered the 'gold standard', for studying ion channel function and pharmacology. While systems offering the luxury of automation have only been commercially available for two decades, the road leading to this new technology is long and rich in seminal, hands-on, studies dating back as far as the 18th century. So where does this technology currently stand, and what will it look like in the future? In the current article, we review the scientific history leading to the development of APC systems, examine key drivers in the rapid development of this technology (such as failed ion channel programmes and the issue of drug-induced hERG inhibition and QT interval prolongation), highlight key capabilities and finally provide some perspective on the current and future impact of the technology on cardiac safety assessment and biomedical science.

Research paper thumbnail of Advances in ion channel high throughput screening: where are we in 2023?

Expert Opinion on Drug Discovery, Dec 17, 2023

Research paper thumbnail of News and views on ion channels in cancer: is cancer a channelopathy?

Frontiers in Pharmacology, Nov 13, 2023

Research paper thumbnail of Adventures and Advances in Time Travel With Induced Pluripotent Stem Cells and Automated Patch Clamp

Frontiers in Molecular Neuroscience

In the Hollywood blockbuster “The Curious Case of Benjamin Button” a fantastical fable unfolds of... more In the Hollywood blockbuster “The Curious Case of Benjamin Button” a fantastical fable unfolds of a man’s life that travels through time reversing the aging process; as the tale progresses, the frail old man becomes a vigorous, vivacious young man, then man becomes boy and boy becomes baby. The reality of cellular time travel, however, is far more wondrous: we now have the ability to both reverse and then forward time on mature cells. Four proteins were found to rewind the molecular clock of adult cells back to their embryonic, “blank canvas” pluripotent stem cell state, allowing these pluripotent stem cells to then be differentiated to fast forward their molecular clocks to the desired adult specialist cell types. These four proteins – the “Yamanaka factors” – form critical elements of this cellular time travel, which deservedly won Shinya Yamanaka the Nobel Prize for his lab’s work discovering them. Human induced pluripotent stem cells (hiPSCs) hold much promise in our understandi...

Research paper thumbnail of Novel oral voltage-gated sodium channel (Na v ) inhibitors inhibit capsaicin induced cough in conscious guinea pigs

European Respiratory Journal, 2015

Rationale: Intractable chronic cough is a serious unmet clinical problem and new therapies are ne... more Rationale: Intractable chronic cough is a serious unmet clinical problem and new therapies are needed for this condition. Na v channels are key for nerve conduction. Local anesthetics that are known to block all Na v channels are effective antitussive agents but their non-selectivity precludes their regular use in patients due to their side effects. Aim and Objectives: Our aim was to compare two novel and selective oral Na v inhibitors vs. a non-selective reference Na v inhibitor in an animal cough model. Methods: Compound potency in Na v channels was assessed using a IonWorks Quattro Automated Patch Clamp System (IC50). Cough was induced in conscious guinea pigs by exposure to aerosolized capsaicin (30µM) and cough numbers were recorded. Results: Compound A showed a dual Na v 1.7-1.8 selectivity with IC50 of 0.5 and 2.6µM, respectively, while compound B was a Na v 1.7 selective compound (IC50 0.07µM). Both compounds inhibited the capsaicin induced cough at similar levels than the pan Na v inhibitor GSK 2338345 (compound A 70%; Compound B 60%; GSK 2338345 71% at 30mg/kg p.o.). Conclusions: These two novel selective oral Na v inhibitors from two distinct chemical series are able to significantly reduce capsaicin induced cough in guinea pigs at similar levels than the non-selective reference compound GSK 2338345. Selective oral Na v inhibitors may be a valuable therapy for chronic cough with lower potential for adverse effects.

Research paper thumbnail of On the perspective of an aging population and its potential impact on drug attrition and pre-clinical cardiovascular safety assessment

Journal of Pharmacological and Toxicological Methods

Research paper thumbnail of Electrophysiological investigations into the G-protein modulation of the neuronal L-type (alpha1D, Cav1.3) and N-type (alpha1B Cav2.2) voltage dependent calcium channels

Doctoral thesis, UCL (University College London)., 2000

Research paper thumbnail of Changes in Local S4 Environment Provide a Voltage-sensing Mechanism for Mammalian Hyperpolarization–activated HCN Channels

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function ... more The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH 2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitution...

Research paper thumbnail of Advancing Ion Channel Research with Automated Patch Clamp (APC) Electrophysiology Platforms

Ion Channels in Biophysics and Physiology, 2021

Since its development on the cusp of the new millennium, automated patch clamp (APC) technology h... more Since its development on the cusp of the new millennium, automated patch clamp (APC) technology has matured over the last two decades. The increased throughput it afforded promised a new paradigm in ion channel recordings: It offered the potential to overcome the time-consuming, low-throughput bottleneck arising from manual patch clamp (MPC) investigations. This chapter highlights the advances in technology, showing how APC platforms have 'democratised' ion channel recordings, lowering the technical bar whilst substantially raising throughput. It will describe the background of the seminal first-generation and updates on advances in second-generation platforms. Furthermore, the chapter summarises the advances APC has made in ion channel studies, including finding new tool compounds and medicines. New functionality and applications on APC platforms give ion channel researchers flexible tools to study ion channels with high quality and high throughput.

Research paper thumbnail of Calcium Channel b Subunit Promotes Voltage-Dependent Modulation of a 1 B by G bg

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming a1 subu... more Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming a1 subunit and auxiliary subunits, including the intracellular b subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free Gbg dimer concentration. Here we have examined the interaction between the regulation of N-type (a1B) channels by their b subunits and by Gbg dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC b subunit, we found a significantly larger Gbg-dependent inhibition of a1B channel activation when the VDCC a1B and b subunits were coexpressed. In the absence of coexpressed VDCC b subunit, the Gbg dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channe...

Research paper thumbnail of Use of automated patch clamp in cardiac safety assessment: Past, present future perspectives

Journal of Pharmacological and Toxicological Methods

Research paper thumbnail of Pain-Related Ion Channel Research : Insights from Industry and Academia

Automated patch clamp (APC) technology was first developed at the turn of the millennium. The inc... more Automated patch clamp (APC) technology was first developed at the turn of the millennium. The increased throughput it afforded promised a new paradigm in ion channel recordings: it offered the potential to overcome the time-consuming, lowthroughput bottleneck arising from manual patch clamp (MPC) investigations. This has relevance to the fast-paced development of novel therapies for chronic pain. This review highlights the advances in technology, using select examples, that have facilitated APC usage in both industry and academia. It covers both first generation and the latest developments in second-generation platforms. In addition, it also provides an overview of the pain research field and how APC platforms have furthered our understanding of ion channel research and the development of pharmacological tools and therapeutics. APC platforms have much to offer the ion channel research community and this review highlights areas of ‘best practice’ for both academia and industry. The i...

Research paper thumbnail of Generating Potent and Selective Inhibitors of Kv1.3 Ion Channel by Fusing Knottins (Venom Derived Mini Proteins) into Peripheral CDR Loops of Antibodies

Biophysical Journal

surface to a large cytoplasmic cavity, and a unique configuration of the photoactive site likely ... more surface to a large cytoplasmic cavity, and a unique configuration of the photoactive site likely responsible for the delayed Schiff base deprotonation. Using this structural information, we have identified residues that determine absorption wavelength and current-voltage dependence. Our results provide clues for rational engineering of ACR molecules further to increase their optogenetic utility.

Research paper thumbnail of Knotbodies: A New Generation of Ion Channel Therapeutic Biologics Created by Fusing Knottin Toxins into Antibodies

Biophysical Journal

and perinexal regions. We correlated nano-structural organisation of junctions to their function,... more and perinexal regions. We correlated nano-structural organisation of junctions to their function, as niches for Na v 1.5. Scanning ion conductance microscopy resolved junctional topography in neonatal rat ventricular cardiomyocyte. This was combined with cellattached Na v 1.5 recordings from cardiomyocyte-cardiomyocyte and cardiomyocyte-fibroblasts junctions. Functional and dominant-negative Cx43 EGFP adenoviruses were used to probe active/inactive junctions. The Na v b1 adhesion inhibitor peptide (badp1) was tested. Whole-cell Na v -currents were recorded to separate non-junctional badp1 effects. To assess transdepolarisation, current-clamp and optical measurements were combined. Plasma membranes were analysed for junctional protein density. Exogenously-delivered Cx43 EGFP trafficked to regions where Na v activity and cluster size were increased relative to native Cx43-junctions (p=0.015). Smaller Na v -clusters (5-10) were abundant at native junctions, larger clusters (20-30) predominated at Cx43 EGFP -junctions. Acute badp1 peptide treatment caused Na v downregulation at native (p<0.01) and Cx43 EGFP -rich junctions

Research paper thumbnail of Using automated patch clamp electrophysiology platforms in pain-related ion channel research: insights from industry and academia

British Journal of Pharmacology

Automated patch clamp (APC) technology was first developed at the turn of the millennium. The inc... more Automated patch clamp (APC) technology was first developed at the turn of the millennium. The increased throughput it afforded promised a new paradigm in ion channel recordings: it offered the potential to overcome the time-consuming, lowthroughput bottleneck arising from manual patch clamp (MPC) investigations. This has relevance to the fast-paced development of novel therapies for chronic pain. This review highlights the advances in technology, using select examples, that have facilitated APC usage in both industry and academia. It covers both first generation and the latest developments in second-generation platforms. In addition, it also provides an overview of the pain research field and how APC platforms have furthered our understanding of ion channel research and the development of pharmacological tools and therapeutics. APC platforms have much to offer the ion channel research community and this review highlights areas of 'best practice' for both academia and industry. The impact of APC platforms and the prospects for chronic pain ion channel research and improved therapeutics will be evaluated.

Research paper thumbnail of Probing S4 and S5 segment proximity in mammalian hyperpolarization-activated HCN channels by disulfide bridging and Cd2+ coordination

Pflügers Archiv - European Journal of Physiology, 2009

We explored the structural basis of voltage sensing in HCN1 hyperpolarization-activated channels ... more We explored the structural basis of voltage sensing in HCN1 hyperpolarization-activated channels by examining the relative orientation of the voltage-sensor and pore domains. The opening of channels engineered to contain single cysteine residues at the extracellular ends of the voltagesensing S4 (V246C) and pore-forming S5 (C303) domains is inhibited by formation of disulfide or cysteine:Cd 2+ bonds. As Cd 2+ coordination is promoted by depolarization, the S4-S5 interaction occurs preferentially in the closed state. The failure of oxidation to catalyze dimer formation, as assayed by Western blotting, indicates the V246C:C303 interaction occurs within a subunit. Intriguingly, a similar interaction has been observed in depolarization-activated Shaker Kv channels at depolarized potentials but such an intrasubunit interaction is inconsistent with the X-ray crystal structure of Kv1.2, wherein S4 approaches S5 of an adjacent subunit. These findings suggest channels of opposite voltage-sensing polarity adopt a conserved S4-S5 orientation in the depolarized state that is distinct from that trapped upon crystallization.

Research paper thumbnail of ω-conotoxin MVIIC reversibly inhibits a human N-type calcium channel and calcium influx into chick synaptosomes

Neuropharmacology, 1994

ABSTRACT

Research paper thumbnail of Changes in Local S4 Environment Provide a Voltage-sensing Mechanism for Mammalian Hyperpolarization-activated HCN Channels

The Journal of General Physiology, 2003

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function ... more The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions...

Research paper thumbnail of Calcium Channel β Subunit Promotes Voltage-Dependent Modulation of α1B by Gβγ

Biophysical Journal, 2000

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming ␣1 subu... more Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming ␣1 subunit and auxiliary subunits, including the intracellular ␤ subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G␤␥ dimer concentration. Here we have examined the interaction between the regulation of N-type (␣1B) channels by their ␤ subunits and by G␤␥ dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC ␤ subunit, we found a significantly larger G␤␥-dependent inhibition of ␣1B channel activation when the VDCC ␣1B and ␤ subunits were coexpressed. In the absence of coexpressed VDCC ␤ subunit, the G␤␥ dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC ␤ subunit coexpression restored all of the hallmarks of G␤␥ modulation. These results suggest that the VDCC ␤ subunit must be present for G␤␥ to induce voltage-dependent modulation of N-type calcium channels.