Toiling in between channels and transporters
David C. Gadsby, Ph.D.
March 26, 1947 to March 10, 2019
David C. Gadsby, Ph.D. passed away in New York City on March 10, 2019. Dr. Gadsby was a Professor Emeritus at Rockefeller University, and had been Associate Editor of the Journal of General Physiology for 24 years, from 1984 to 2008, as well as president of the Society of General Physiologists between 1999 and 2000. He was the 1995 recipient of the K. S. Cole Award of the Biophysical Society, and a member of the Royal Society since 2005.
Following completion of his PhD at University College London, Dr. Gadsby joined Paul Cranefield's laboratory at the Rockefeller University, New York, in 1975. Studying electrical properties of cardiac Purkinje fibers, he developed methods to selectively measure the small currents carried by the electrogenic transport cycle of the Na+/K+ pump. Becoming quickly independent, he continued his research on the pump's transport mechanism, pioneering the application of the patch-clamp technique for studying currents of a transporter. Using systematic quantitative analysis he showed that the Na+/K+ pump reaction cycle includes only a single voltage-dependent step which, however, does not limit the rate of either the forward or the reverse transport cycle. He later identified K+ translocation as the rate-limiting step, and measured rate constants for all the individual steps of the pump cycle.
In 1989, David Gadsby, Robert Rakowski, and Paul De Weer developed a revolutionary system which allowed simultaneous measurement of Na+/K+ pump current and ion flux from a controlled membrane area of a squid giant axon perfused both internally and externally. This started a two-decade long annual adventure of the trio, later joined by Francisco Bezanilla and Miguel Holmgren, at the Woods Hole Marine Biological Laboratory in Massachusetts, exploiting the squid season for advancing the fundamental understanding of Na+/K+ pump function. Using this system the team identified that the release of external Na+ (or its rebinding in reverse-mode operation) is the predominant charge-moving step in the pump's cycle, suggesting that extracellular Na+ ions must reach their binding sites deep in the pump molecule through a high-field access channel. This finding implied that part of the pump molecule is functionally analogous to an ion channel. Using high-speed voltage jump experiments they were later able to clearly show that during normal-mode cycling of the pump the three transported Na+ ions are de-occluded and released to the extracellular solution one at a time, in a strict order.
Consistent with idea that the extracellular parts of the pump may form an ion-channel-like structure, the marine toxin palytoxin was found to convert Na+/K+ pumps into nonselective cation channels by disrupting the normal strict coupling between opening of one access pathway and closing of the other. Studying regulation of gating of single palytoxin-bound Na+/K+ pump-channels by the physiological ligands ATP, Na+, and K+, Gadsby’s lab was first to demonstrate that, despite the bound palytoxin, partial reactions of the normal pump cycle persist. This affirmed the alternating access model of ion pumps, and allowed the study of ion occlusion and deocclusion steps at the microscopic level, in single Na+/K+ pump molecules. Using cysteine scanning of the anticipated ion permeation pathway in palytoxin-bound Na+/K+ pump-channels, their studies discovered not only a wide but deep outer vestibule which leads to a narrow charge-selectivity filter formed by acidic residues that allow the approach of cations but exclude anions, but also a single unbroken cation pathway that traverses palytoxin-bound Na+/K+ pump-channels from one side of the membrane to the other. In recent years, the lab’s work identified inward proton transport through the actively cycling Na+/K+ pump, classifying it among hybrid channel-transporters. The potential pathological relevance of pump-mediated proton influx during extracellular acidification remains to be explored.
In 1989, the year the gene cftr, loss-of-function mutations of which cause cystic fibrosis, was cloned, Dr. Gadsby discovered a cAMP-dependent chloride current in cardiac myocytes. Investigating its regulation via G proteins, as well as its single-channel properties, he later concluded that this cardiac chloride current is carried by the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an epithelial chloride ion channel known to be activated through phosphorylation by cAMP-dependent protein kinase, and gated by binding and hydrolysis of cytosolic ATP. That discovery started off a second line of decades-long research in his lab, focused on understanding structure and function of CFTR, the only channel member in the ABC transporter family, whose failure causes cystic fibrosis. Using non-hydrolyzable ATP analogs, vanadate, or Mg2+ removal to disrupt the ATP hydrolysis cycle at CFTR's two cytosolic nucleotide binding domains (NBDs) he identified strong coupling between ATP hydrolysis and channel closure, and started to dissect how incremental phosphorylation of its unique cytosolic regulatory domain affects the mechanism of channel gating. To address how structural manipulations affect CFTR channel function, research in the Gadsby lab switched from cardiac myocytes to the frog oocyte heterologous expression system. Exploiting split CFTR channel constructs, obtained by coexpression of its two homologous halves, their studies could demonstrate strong functional asymmetry between the two ATP binding sites. Whereas binding of ATP at NBD2 leads to channel opening and its hydrolysis there prompts channel closing, at NBD1 ATP remains tightly bound and unhydrolyzed for periods that are very long compared to the channel's gating cycle. Emerging structural information on related ABC proteins, together with sequence comparisons across the entire ABC family, allowed identification of key residue interactions that change in a gating-state dependent manner. Using thermodynamic mutant cycles work in the Gadsby lab showed that opening of the CFTR channel pore is coupled to the formation of a stable intramolecular NBD1-NBD2 heterodimer following ATP binding, and that pore closure is coupled to disruption of that dimer, triggered by ATP hydrolysis. These key observations, later confirmed by state-dependent cross-linking of pairs of engineered cysteines, allowed him to explain how the conserved mechanism of the ABC-family active transport cycle is exploited in CFTR to drive opening and closure of the ion channel pore, and suggested that CFTR evolved by degeneration of the intracellular ABC transporter gate. Atomic structures of CFTR obtained a decade later have largely confirmed those predictions.
In addition to his outstanding scientific accomplishments, Dr. Gadsby has trained a large number of young scientists who continue to carry on his work on both channels and pumps. As a mentor, he was committed to passing on to the younger generation his depth and clarity of thinking, his uncompromising rigor in experimentation as well as data interpretation, and his insatiable curiosity towards deeper understanding of natural phenomena. At the same time, he granted great freedom to young scientists, allowing them to delve into problems that attracted their attention, and encouraged them to fully enjoy the excitement of scientific discovery. He showed exceptional patience and understanding, and was not only respected but truly loved by his trainees. Outside the workplace, Dr. Gadsby and his wife Patricia Gadsby cherished friendship with both scientists and non-scientists around the globe. He enjoyed traveling, and was a keen fisherman, often seen cruising the seas around Woods Hole in his boat. All of us who have had the chance to know him feel extremely grateful for this privilege.
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