Cryo-EM technology helps elucidate KATP channel pharmacology, with implications for drug development

Shyng Lab, research team

In the human body, the proper release of insulin maintains healthy levels of glucose (sugar) in the blood. When blood glucose levels rise following the intake of food, pancreatic β-cells release insulin that instruct the body to use or store excess glucose, thus maintaining blood glucose in a healthy range. Insufficient insulin secretion results in high blood sugar, a condition known as diabetes; uncontrolled insulin secretion results in low blood sugars.

The ability of β-cells to increase their output of insulin as food is digested is based on a remarkable energy-sensitive ion channel, known as the ATP-sensitive potassium (KATP) channel, located in the cell surface membrane, explains Show-Ling Shyng, Ph.D., professor of chemical physiology and biochemistry, OHSU School of Medicine. “These channels are regulated by the glucose metabolites, ATP and ADP, enabling them to couple blood glucose levels with membrane excitability, resulting in insulin secretion.”

If mutations in the channel’s genes impair the production process of the channel, the result is a severe form of an insulin over-secretion disease known as congenital hyperinsulinism. The current treatment for it typically requires surgical removal of the pancreas to prevent life-threatening hypoglycemia.

“Better pharmacological treatment options would greatly improve the quality of life for patients with this devastating disease,” said Dr. Shyng. “However, our limited understanding of the mechanisms that govern channel biogenesis in physiological and pathological states has hampered significant progress.”

Dr. Shyng’s team set out to answer this question. The resulting study, “Mechanism of pharmacochaperoning in a mammalian KATP channel revealed by cryo-EM” published in eLife was selected as the School of Medicine’s Paper of the Month.

Power of cryo-EM

Previous studies over the last 15 years identified several small molecules which “rescue” trafficking-impaired mutant KATP channels to the cell surface, explains Dr. Shyng. These compounds, referred to as pharmacological chaperones, include well-known KATP channel-targeting drugs – sulfonylureas and glinides – widely used to treat type 2 diabetes, as well as carbamazepine, an anticonvulsant previously thought to target primarily voltage-gated sodium channels.

“How these structurally dissimilar compounds correct mutant channel trafficking defects had remained enigmatic,” said Dr. Shyng. “A major obstacle in understanding the underlying mechanisms was the lack of high-resolution channel structures.”

But, using OHSU’s single-particle cryoEM imaging technology, the team obtained several channel structures at near-atomic resolutions.

“In particular, we have resolved the binding site of several drugs known to act as pharmacological chaperones of the channel,” said Dr. Shyng.

A common link

In the study, the team determined cryoEM structures of the channel, composed of two subunits, Kir6.2 and SUR1, in the presence of three distinct pharmacochaperones, glibenclamide (GBC, a sulfonylurea), repaglinide (RPG, a glinide), and carbamazepine (CBZ).

“We showed that all three drugs occupy the same binding pocket within the SUR1 subunit. Further, using cross-linking combined with mass spectrometry and electrophysiology mapping approaches, we showed that the distal Kir6.2 N-terminus is trapped in the cavity formed by the two halves of the SUR1 core structure next to the drug binding pocket,” said Dr. Shyng. “The distal Kir6.2 N-terminus is known to be critical for channel assembly and has been shown to contribute to drug binding. Our results suggest the different small molecules correct the assembly and trafficking defects of mutant channels by stabilizing a key regulatory interaction between the N-terminus of Kir6.2 and SUR1.”

The study provides insight into the structural mechanism by which SUR1 and Kir6.2, two evolutionarily and structurally distant proteins, form a functional unit to sense metabolic sensors.

“It helps us understand how disease mutations disrupt channel assembly and how drugs help stabilize specific interactions to overcome mutation-induced defects,” said Dr. Shyng. “We can now explain decades of pharmacological and structure-function data. Moreover, the structures reveal in unprecedented details how these drugs interact with the channel.”

She added, “With this knowledge, structure-guided drug design is now possible to improve existing drugs and the development of new therapeutic drugs, not only for congenital hyperinsulinism but also for neurological disorders caused by KATP mutations as well as type 2 diabetes, which is one of the most serious health problems we face today.”

Looking ahead

“What I found fascinating about this study was the team’s insight into a common mechanism for multiple drug classes,” said Mary Heinricher, Ph.D., associate dean for research, OHSU School of Medicine.

Looking ahead, Dr. Shyng’s team hopes to further improve the resolution of their structures and use them as a drug development platform.

“KATP channels composed of other SUR and Kir isoforms are known to be expressed in heart, smooth muscle, skeletal muscle and the brain where they have important functions, including control of vascular tone and the protection of cells against ischemic injury,” said Dr. Shyng. “We are interested in solving structures of additional KATP channel subtypes with the goal of developing subtype-specific drugs for targeted therapies.”

Importance of OHSU Research Cores and Shared Resources

These findings were possible thanks to OHSU cores. Dr. Shyng would like to acknowledge the following:

Pictured above: Dr. Craig Yoshida, Dr. Show-Ling Shyng, Zhongying Yang, Dr. Larry David, Dr. Min Woo Sung. Not pictured: Dr. Gregory Martin.