pH sensing protein reveals two new ways for cells to detect proton

Specialized cells in our bodies utilize protein nanosensors to regulate essential processes including our metabolism, heart rate, and breathing. Below, biophysics graduate student Robert Rietmeijer describes high-resolution reconstructions of the pH sensing ion channel TASK2, and how natural nanosenors like TASK2 work.

Your body uses precise sensors, switches, and gates to constantly surveil and react to your surroundings. Even seemingly passive processes like the breath you just took would not be possible without this precise and delicate molecular machinery. However, scientists are only beginning to understand how these natural nanosensors work.

Researchers at UC Berkeley have determined how one such nanosensor, the pH sensing protein TASK2, senses changes in pH and generates an electrical signal. Whereas electronic devices use currents of electrons, cells produce and respond to currents of sodium, potassium, calcium, and chloride ions. Proteins called ion channels are embedded in cell membranes and act as nanoscopic gatekeepers, opening or closing to control the flow of these ions. Like gatekeepers, ion channels require different “passwords,” to open and close. In lieu of actual words, ion channels can sense environmental conditions, like pH, to determine whether they open or close. TASK2 possesses two pH sensors that enable the channel to cut off the electrical signal it creates, alerting the cell of a potentially fatal increase in acidity.

Two detailed 3D models, reported this week in Nature, show that the pH sensitive ion channel TASK2 has two gates, one for sensing intracellular pH and one for extracellular pH. “Surprisingly, the mechanisms of the extracellular and intracellular gating are both different from the reported mechanisms of other related channels,” remarks Baobin Li, one of the study’s coauthors and a postdoctoral fellow in Steve Brohawn’s lab.

The 3D models may be used as templates for designing drugs that could establish a new avenue for treating breathing disorders. TASK2 is the pH sensing ion channel that neurons in the brainstem use to regulate the rate of respiration (by measuring CO2 dissolved into our blood as carbonic acid) and proximal tubules in the kidneys use to efficiently recycle salts.  “As a potential drug target, there is no available specific inhibitor or activator of TASK2,” Li notes. “This structural information of TASK2 in closed and open states paves the ways for computer-aided drug design to develop specific activators or inhibitors.”

Nature’s Nanosensors

Ion channels like TASK2 use gates to convert information about the environment into an electrical signal—a current of ions flowing into or out of the cell. These gates function simultaneously as sensors and switches that translate input signals, like pH, into an electrical current. They do this by coupling a pore, through which ions can freely flow, to a gate that blocks the flow of ions when a condition, like a threshold pH value, is not met. This simple open/closed scheme even allows these channels to encode the magnitude of the pH change as the magnitude of the current it passes. By modulating the frequency and duration of the open and closed states these channels can pass little or no current at pH lower than 6.5, more current at pH 7 or 8, and a lot of current at pH greater than 9.

The nanoscopic gating in ion channels is comparable to the macroscopic gates we encounter in everyday life: portals close, corks plug, airlocks seal, valves pinch.

The extracellular gate appears to pinch closed to block the flow of ions in a novel way. “Movement of an extracellular facing arginine residue results in a series of coordinated rearrangements that induce the collapse of the pore,” observes Li. “The consequent loss of two potassium coordination sites disfavors the conduction of ions through the channel.”

Protonation of a distant arginine (ARG 224) residue causes a series of rearrangements (ARG 224, ASN 87, ASN 82, V104, G102) that pinch the potassium selective pore of the channel closed by eliminating two potassium coordination sites.

Comparing the 3D reconstructions of the open and closed channels reveals a second gate on the intracellular side of the channel that closes more like an airlock. “Different from other channels, protonation of an intracellular facing lysine residue frees it to twist ninety degrees and create a seal that blocks the passage of potassium ions,” Li notes.

Protonation of a lysine (LYS 245) residue allows it to rotate and seal the intracellular gate at low pH.

While the general location of these gates had been known for more than a decade, these structures reveal the unique chemical environments that enable the precise pH sensitivity of TASK2. The researchers used the structures to discover new amino acid residues critical to either the sensitivity of the pH sensor or the integrity of the gate, information that could help scientists design small molecules that stabilize either gate in an open or closed state. These drugs could already be useful in the clinic but would also provide researchers powerful new tools for uncovering yet undiscovered physiological roles of TASK2.

Multitasking Molecules

TASK2 is a member of a family of potassium channels, the K2P family, that gate in response to extremely diverse stimuli. “K2P channels help maintain the resting membrane potential of cells and regulate their electrical excitability. The activity of K2P channels can be regulated by mechanical force, pH, heat, and signaling molecules like polyunsaturated fatty acids and G proteins,” says Li.

Each family member responds to two or more of these stimuli, which means that these channels are packed with sensors and gates, functioning as nanoscopic multi-meters to inform the cell about its environment. In addition to sensing pH, TASK2 also senses the signaling molecule PIP2, a phospholipid that may tune the pH sensitivity of TASK2 according to the developmental or metabolic state of the cell. The structural basis of this sensitivity is not obvious from the 3D structures in this study, and constitutes an exciting follow up question: Does PIP2 hijack the intracellular pH gate, or does the channel possess an undiscovered third gate that specifically senses PIP2?

In nearly every organ in the body, cells employ this 15-member family of multitasking molecules as sensors, including the central and peripheral nervous systems, heart, epithelia, kidneys, and pancreas. In each of these tissues, highly specialized cells use these channels to sense rapid physical and chemical changes and encode them into an electrical signal for the cell to act on. “The 15 different K2P channels in mammals play important roles in a wide number of physiological systems,” says Dr. Li. Consequently, these channels are implicated numerous diseases, “K2P channels are involved in the regulation of heart rate, sleep/wake cycle, regulatory cell volume decrease, bicarbonate reabsorption, breathing control and CO2 homeostasis, diabetes, depression, pain, and anesthesia.”

K2P channel pharmacology, however, is a nascent field with no approved drugs that specifically activate or inhibit K2P channels, but at least two in clinical trials. 3D structures of K2P channels such as the ones of TASK2 reported here provide key resources for developing drugs that will specifically target these channels. High-resolution 3D structures provide researchers with maps that often reveal where interesting functional regions of the protein are and can show where candidate drugs bind the protein. Having a high-resolution structure of a molecule is kind of like looking at the opponent’s board in a game of battleship—you start the game with a good idea of where the interesting “hits” will be.