At Stanford University School of Medicine, Merritt Maduke, Ph.D., assistant professor in the department of molecular and cellular physiology, has built a lab focused on elucidating the molecular mechanisms underlying the function of the CLC chloride channels, the largest molecular family of mammalian anion channels.
There are nine mammalian members, CLC-1 to 7 and Ka and Kb for the kidney-specific type. Four family members are expressed on the plasma membrane of various cell types; five are expressed on intracellular membranes. Dr. Maduke’s interest in the CLC family is that half of the members are chloride channels, while the other half are chloride-proton antiporters.
“Historically, ion channels and antiporters have been thought of as two distinct classes of membrane proteins. Channels allow only passive movement of ions; antiporters can be used to pump ions. The finding of both of these classes of membrane proteins within a single gene family means that the mechanisms are likely not as distinct as had been previously supposed,” indicated Dr. Maduke. “Study of these mechanisms will help us understand how similar genes have evolved to carry out these different functions and will thus provide fundamental insight into how membrane proteins work.”
The CLC-1 channels found in skeletal muscle are closed at rest and upon excitation, i.e., membrane depolarization, the chloride channels open allowing chloride to enter into the cell. The negative charge entering in repolarizes the membrane, which allows the muscle to relax. Mutations in the CLC-1 channels cause myotonia in humans, a state where fewer CLC channels open, impairing membrane repolarization and muscle relaxation. Individuals with myotonia have problems with activities that involve skeletal muscle.
The physiological role for CLC-7 antiporters, one of the members expressed on intracellular membranes, is quite interesting. In osteoclasts, CLC-7 channels are localized to a specialized membrane called the ruffled border. Osteoclasts adhere to the bone surface and secrete protons and hydrolytic enzymes to degrade and absorb the attached bone.
Throughout our lives, bone is continually chewed up by osteoclasts and built up by osteoblasts. CLC-7 is essential for this process because it allows chloride to also be secreted into the bone matrix. This also balances the charge built up from proton secretion, enabling further proton movement.
Since CLC-7 is essential for the process of bone degradation, the prevailing thought is that by knocking down CLC-7 function one would limit the ability of osteoclasts to degrade bone.
For Dr. Maduke, the question that keeps her focused is how are the mechanisms of CLC chloride channels and CLC chloride/proton antiporters related. To get at this answer, x-ray crystallographic structural analysis of the CLC-ec1 homolog in E. coli provides some basic information. But this data only gives a single conformational view of the protein.
Dr. Maduke and her team are looking to understand how the proteins move as they open and close to allow regulated flux of ions. The team is looking to NMR and deuterium exchange detected by mass spec to obtain snapshots of the proteins in action.