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Jun 1, 2008 (Vol. 28, No. 11)

Ion Channels Play Key Role in R&D

Subtype Selectivity Is Being Developed to Improve Efficacy and Reduce Side Effects

  • Maintenance of cardiac rhythm and pain sensation require the flow of ions across the membranes of all cells. Ion channels, which facilitate this movement, are protein assemblies that penetrate the membrane and catalyze passage of specific ions such as sodium (Na+), potassium (K+), or calcium (Ca2+) into or out of the cell down the electrochemical gradient.

    Ion channels are as diverse as they are numerous (more than 400 channel genes have been described) and they are grouped into different families based on which specific ion flows through them and how they are gated. That is whether they open or close in response to a chemical (ligand gated), electrical signal (voltage gated), pH, temperature, or mechanical force.

    Ion channels play a role in a wide variety of biological processes that involve rapid signaling such as neural signal transduction; cardiac, skeletal, and smooth muscle contraction; epithelial transport of nutrients and ions; T-cell activation; and pancreatic beta-cell insulin release. It is not surprising then that ion channels are favorite targets of pharmaceutical companies and also hot topics at recent and upcoming conferences including Select Biosciences’ “Ion Channel Targets” and “Target Opportunities”, and Informa’s “Ion Channel Targets.”

  • Basic Biology

    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.

  • Mechanosensitive Channels

    Frederick Sachs, Ph.D., distinguished professor at the Center for Single Molecule Biophysics in the physiology and biophysical sciences department at SUNY Buffalo, is working on perhaps the oldest ion channel.

    Dr. Sachs’ serendipitous discovery of mechanosensitive ion channels (MSC) in skeletal muscle led to experiments linking cell mechanics and the mechanisms of mechanical signal transduction. The Sachs lab uses patch-clamp, high-resolution microscopy, molecular biology, and structural NMR to study these ion channels.

    MSCs are ubiquitous. While most familiar in the sensory cells of touch and hearing, they are found in a similar density in all cells. Probably arising as a method to balance cell volume under the osmotic drive of metabolism, they represent an evolutionary history from bacteria to humans. As opposed to many other channels, their activity is not regulated by channel density but by regulating the mechanical shielding of the channels by the cytoskeleton.

    The Sachs lab has discovered a specific inhibitor of MSC—GsMtx-4, a small peptide isolated from the venom of Grammostola spatulata, a tarantula. The tarantula peptide has a unique pharmacology since the D- and the L-forms are equally active, but the site of action is specific to cation-selective mechanosensitive ion channels.

    The peptide may have significant clinical applications since it inhibits cardiac arrhythmias, calcium influx in dystrophic muscle, spontaneous channel activation (and hence contraction) of human bladder smooth muscle, and suppresses stretch-induced endothelia production by glial cells that may play a role in brain tumor development. Furthermore, the peptide is nontoxic in mice.

    “I have a recurring pharmaceutical dream for the millions of Americans with atrial fibrillation,” Dr. Sachs said. “If we provide the peptide in an inhaler when a patient feels an atrial fibrillation episode coming on, they can take a hit on their inhaler to stop the acute attack.

    “Since atrial fibrillation begets atrial fibrillation, acute inhibition should improve long-term prospects for the patient. Since the peptide doesn’t affect the ion channels responsible for the normal cardiac-action potential such as Kv channels, there are minimal side effects. The peptide only acts on channels activated by abnormal mechanical stress such as overinflation of the atria. And with acute dosing toxicity is minimized.”

    The dream aside, despite their ubiquity, the breadth of knowledge about MSCs, and the availability of a chemically synthesized, specific inhibitor, pharma companies have shied away from tackling this new array of targets, perhaps from the uncertainty of utilizing unfamiliar stimuli.

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