Adventures and Advances in Time Travel With Induced Pluripotent Stem Cells and Automated Patch Clamp.

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 understanding of physiology and medicine. They encapsulate the signaling pathways of the desired cell types, such as cardiomyocytes or neurons, and thus act as model cells for defining the critical ion channel activity in healthy and disease states. Since hiPSCs can be derived from any patient, highly specific, personalized (or stratified) physiology, and/or pathophysiology can be defined, leading to exciting developments in personalized medicines and interventions. As such, hiPSC married with high throughput automated patch clamp (APC) ion channel recording platforms provide a foundation for significant physiological, medical and drug discovery advances. This review aims to summarize the current state of affairs of hiPSC and APC: the background and recent advances made; and the pros, cons and challenges of these technologies. Whilst the authors have yet to finalize a fully functional time traveling machine, they will endeavor to provide plausible future projections on where hiPSC and APC are likely to carry us. One future projection the authors are confident in making is the increasing necessity and adoption of these technologies in the discovery of the next blockbuster, this time a life-enhancing ion channel drug, not a fantastical movie.

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

There is no refuting that America's population is growing older: for the first time in US history, by 2034 older adults (defined as >65 years of age) are projected to outnumber children under the age of 18, representing approximately 70 million people or almost 25% of the population (Lloyd-Jones et al., 2010). Described as the "silver tsunami", this flood of older adults is driven by the baby boomers (people born after World War II, from 1946 to 1964): they are now reaching old age, living longer due to significant advances in healthcare coupled with a record low birth rate, resulting in a skewed elderly population demographic. Unfortunately, older adults are also becoming increasingly unhealthy. Many often suffer from several chronic disorders requiring the use of multiple medications at a level higher than any other age group, resulting in an increased risk of drug-drug interactions (DDIs) and adverse drug reactions (ADRs). Indeed, because of age-related changes in pharmacokinetics (PK) and pharmacodynamics (PD), older adults are also more vulnerable to drug toxicity. Prescribed drugs certainly improve a range of health outcomes, but also often cause considerable ADRs, leading to devastating consequences for patients, clinicians, and manufacturers. Therefore, safe and effective pharmacotherapy remains one of the greatest growing challenges in geriatric medicine. In this review we examine the effects of aging and its impact on the increased risk of experiencing ADRs, resulting in devastating consequences for patients and manufacturers. We assess the current regulatory considerations related to the development of drugs for this population and highlight issues, concerns, and propose alternatives to the standard battery of tests focused on assessing cardiovascular (CV) safety in an attempt to develop safer and efficient new drugs for the growing elderly demographic.

Use of automated patch clamp in cardiac safety assessment: Past, present & future perspectives.

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.

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

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.

Advancing Ion Channel Research with Automated Patch Clamp (APC) Electrophysiology Platforms.

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.

Using automated patch clamp electrophysiology platforms in 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 increased throughput it afforded promised a new paradigm in ion channel recordings, offering the potential to overcome the time-consuming, low-throughput bottleneck, arising from manual patch clamp 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 to 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 of ion channel research and improved therapeutics for chronic pain will be evaluated. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Probing S4 and S5 segment proximity in mammalian hyperpolarization-activated HCN channels by disulfide bridging and Cd2+ coordination.

We explored the structural basis of voltage sensing in the HCN1 hyperpolarization-activated cyclic nucleotide-gated cation channel 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 voltage-sensing 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 voltage-dependent potassium (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.

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 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 in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (approximately 3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.