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February 2011

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"Phosphorylopathies," a new class of human disorders

By Negin Martin
February 2011

David Armstrong Ph.D.

Armstrong, an authority on ion channel regulation, was recruited to NIEHS by former Scientific Director Martin Rodbell, Ph.D., in 1988 to establish the Membrane Signaling Group. His research is dedicated to investigating the post-translational regulation of ion channels and the signal transduction pathways that regulate them. (Photo courtesy of Steve McCaw)

On Jan. 4, as part of the Duke University Medical Center's 2010-2011 seminar series on Ion Channel Research, NIEHS Principal Investigator David Armstrong Ph.D., gave a lecture titled "Phosphorylopathies: losing control of ion channel phosphorylation." Armstrong, who is chief of the Laboratory of Neurobiology at NIEHS, presented three examples of aberrant phosphorylation of ion channel proteins that are associated with increased risk of disease.

Inherited mutations in ion channel proteins that disrupt channel formation or ion permeation - the "channelopathies" such as epilepsy and cystic fibrosis - are one of the fastest growing categories of human disease. Channel proteins are not only responsible for the electrical signals in the brain that underlie cognition, but in most tissues, channels are the final molecular switches for maintaining physiological homeostasis. Consequently, their activity must be narrowly regulated, and ion channels have become one of the leading drug targets for pharmacological treatment of human disease.

Armstrong's group employs the patch clamp technique to study ion channels at the molecular level. By recording the picoampere currents through a single channel in the surface membrane of a cell, they can detect every opening and closing of the channel in real time in situ. Recent findings by Armstrong's group highlight the role of protein phosphorylation in regulating ion channels.

Dysregulation of phosphorylation

Reversible protein phosphorylation by kinases, which add, or phosphatases which remove phosphate groups on proteins, is a well-known mechanism for regulating protein function. However, Armstrong and his collaborators have discovered several examples in which dysregulation of phosphorylation, which they have named "phosphorylopathies," leads to loss of control of channel activity and increased susceptibility to disease. Protein phosphatases are also the target of some of the most potent microbial toxins in algal blooms, which also produce dysregulation of channel activity. During his presentation, Armstrong offered examples from his recent research that are related to human health (see text box).

Armstrong said he is pleased with the growing public interest in his field of research. "I enjoyed my visit to the Ion Channel Research Unit in the School of Medicine at Duke University," he noted. "Duke's major investment in this unit reflects the importance of ion channels in maintaining human health." He also noted that a bioinformatics group in China has now assembled a complete list(http://phossnp.biocuckoo.org/index.php) Exit NIEHS of potential phosphorylopathies in the human genome, which is publicly available.

(Negin Martin, Ph.D., is a biologist in the NIEHS Laboratory of Neurobiology Viral Vector Core Facility and a 2009 Science Communication Fellow with Environmental Health Sciences.)

Phosphorylation dysfunction and human health

Immunosuppressants, which prevent lymphocyte activation by inhibiting the calcium-dependent protein phosphatase, calcineurin, are neurotoxic when taken chronically. In collaboration with the NIEHS Transmembrane Signaling Group headed by Lutz Birnbaumer, Ph.D., Armstrong and his group discovered that the voltage-activated CaV1.2 calcium channels encoded by the CaCNA1C gene, which link depolarization to hormone secretion, muscle contraction, and neuronal gene expression, remain open much longer when calcineurin is blocked. The resulting increase in calcium entry is toxic to neurons.

When Armstrong's group identified the amino acid in the CaV1.2 protein that is dephosphorylated by calcineurin, they discovered that a genetic polymorphism which results in Timothy syndrome, a devastating developmental disorder of brain and heart that was previously unrelated to immune suppression in transplant patients, produces a similar consensus site for the kinase at a topologically related region of the channel protein, thus exacerbating the effect of phosphorylation on channel open duration. Armstrong and his colleagues hope to use their new structural insights into calcium channel function to design drugs, such as the dihydropyridines, which are used to treat heart disease but have neurotoxic side effects that selectively block the long openings.

Building on their discovery that the polymorphism responsible for Timothy syndrome dysregulated channel activity by creating a new phosphorylation site on the channel protein, Armstrong and his group asked a summer intern to conduct a bioinformatics search for other examples in the HapMap database in which a single nucleotide polymorphism, or SNP, results in the creation or removal of a phosphorylation site in the protein. They identified fourteen additional examples where a polymorphism that was already known to be associated with human disease was also predicted to alter channel phosphorylation.

The researchers recently confirmed one of these predictions by showing that regulation of the Kv11.1 potassium channels which are encoded by the hERG gene and regulate action potential duration and frequency in cardiac myocytes, endocrine cells and neurons, reverses the effect of hormonal signaling through phosphatidylinositol 3-kinase (PI3K), a lipid kinase. 

Armstrong's group had discovered previously that thyroid hormone receptors signal though PI3K to regulate Kv11 channels in pituitary cells, and they do this by stimulating a different protein phosphatase, PP5. However, PI3K is a central cellular regulator of metabolism and growth, and one of the polymorphisms in the hERG gene created a new site for a PI3K stimulated protein kinase. Consequently instead of hormonal signaling dephosphorylating and stimulating the channels and relaxing the heart, hormonal signaling now increased phosphorylation of the channels and turned them off, which is known to cause cardiac arrhythmias.

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