Cryo-EM Reveals TMEM16A’s Activation Mechanism

Ion channels are proteins in cell membranes that act as molecular gatekeepers. They form pores that open and close to control the flow of ions, a process that is the basis for many biological functions like nerve impulses and muscle contraction. One such gatekeeper is the protein TMEM16A, a member of the anoctamin family. It is a calcium-activated chloride channel, meaning it opens its gate to chloride ions only when prompted by calcium ions inside the cell.

The Role of TMEM16A in Human Health

The TMEM16A protein is widely expressed throughout the human body, contributing to a diverse set of physiological processes by controlling chloride ion movement. In the airways, it is involved in mucus secretion, a process that helps keep our lungs clear. Its function is also observed in the regulation of smooth muscle contraction, which affects blood pressure and airflow.

This protein’s activity extends to controlling fluid secretion in various epithelial tissues, which are the linings of organs and glands. In the digestive system, it participates in the secretion of fluids necessary for digestion. The channel also plays a part in pain signaling within certain neurons.

Given its widespread roles, the dysfunction of TMEM16A is linked to several human diseases. Over-activity of the channel can lead to conditions like hypertension and certain types of secretory diarrhea. Conversely, insufficient function can contribute to the thick, sticky mucus characteristic of cystic fibrosis-like symptoms and may play a role in the airway constriction seen in asthma.

Visualizing the Invisible with Cryo-EM

To understand how a molecular machine like TMEM16A works, scientists must see its structure. For many years, this was a challenge, as proteins are small and dynamic. The development of cryo-electron microscopy (cryo-EM) was a technological leap, allowing researchers to visualize these molecules with new clarity.

The cryo-EM process involves flash-freezing a purified protein sample in a thin layer of vitreous ice, trapping the molecules in their natural state. An electron microscope then captures thousands of two-dimensional images of the frozen, randomly oriented proteins.

These 2D projections are computationally combined using software that aligns the images and reconstructs them into a high-resolution 3D model. This map reveals the molecule’s architecture, showing the arrangement of its atoms and the shape of its components.

This technique was a breakthrough for seeing large proteins like TMEM16A, which were difficult to study using X-ray crystallography. By capturing structures in both the calcium-free (closed) and calcium-bound (open) states, cryo-EM provided direct snapshots of the channel’s activation cycle.

Unveiling the Calcium-Binding Process

The activation of TMEM16A begins with calcium ion binding. Cryo-EM structures pinpointed the location of this binding site, revealing it is nestled within the part of the protein that spans the cell membrane.

A functional TMEM16A channel is a homodimer, formed by two identical subunits. Structural data shows each subunit has its own ion-conducting pore and calcium-binding site. To initiate activation, each subunit must bind two calcium ions in a pocket formed by negatively charged amino acid residues.

This binding site is positioned near the intracellular entrance of the pore. The presence of positively charged calcium ions here alters the electrostatic properties of the ion conduction pathway, which is the trigger for the subsequent opening of the channel gate.

The Structural Shift That Opens the Channel

Once calcium ions bind, the TMEM16A protein undergoes a conformational change. This physical rearrangement is the mechanical action that opens the gate for chloride ions. Cryo-EM structures captured this movement by comparing the protein’s shape in its calcium-free and calcium-bound forms, showing that calcium binding triggers a rearrangement of the transmembrane helices forming the pore.

A key part of this transition involves transmembrane helix 6 (α-helix 6). Upon calcium binding, this helix moves, altering the structure of the pore’s narrowest part, the “neck” region. This shift reconfigures the lining of the ion pathway.

In the closed, calcium-free state, the pore is constricted by the arrangement of the helices. Calcium binding causes α-helix 6 and parts of nearby helices to rotate and shift away from the pore’s central axis. This movement widens the pathway, creating an open channel for chloride ions to pass through.

This transition from a closed to an open state transforms the protein into a conductive channel. The structural details from cryo-EM show how the binding of a small ion causes a large-scale rearrangement and a functional change. This process clarifies how a ligand-gated ion channel operates at a molecular level.

Therapeutic and Research Implications

The 3D maps of TMEM16A in its different states have implications for medicine and research. Having a blueprint of the channel when closed and open allows for rational drug design, where scientists can create molecules shaped to interact with specific parts of the protein.

For diseases caused by an overactive TMEM16A channel, such as hypertension, researchers can design antagonist drugs to block its function. These molecules could target the moving parts of the pore, like α-helix 6, to stabilize the channel in its closed state. Conversely, for conditions where enhancing channel activity is beneficial, molecules could be designed to promote the open state.

This structural knowledge also accelerates basic research. Understanding TMEM16A’s activation mechanism provides insights into the entire anoctamin protein family, which includes members that transport lipids instead of ions. Scientists can now investigate how small structural differences between these related proteins lead to different functions.