Michael C. Puljung

Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

Significance Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play central roles in the heart and the brain. In the heart, they are present in pacemaker cells and contribute to the regulation of the heartbeat. In the brain, they are involved in electrical signaling of neurons. HCN channels are activated by hyperpolarization of the cell membrane and are regulated by binding of cAMP to a site in an intracellular binding domain. This study shows that this binding domain undergoes major structural changes upon binding of cAMP. The results are the first step toward elucidating the molecular mechanism of gating in this important class of ion channels. Binding of 3′,5′-cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates their gating. cAMP binds to a conserved intracellular cyclic nucleotide-binding domain (CNBD) in the channel, increasing the rate and extent of activation of the channel and shifting activation to less hyperpolarized voltages. The structural mechanism underlying this regulation, however, is unknown. We used double electron–electron resonance (DEER) spectroscopy to directly map the conformational ensembles of the CNBD in the absence and presence of cAMP. Site-directed, double-cysteine mutants in a soluble CNBD fragment were spin-labeled, and interspin label distance distributions were determined using DEER. We found motions of up to 10 Å induced by the binding of cAMP. In addition, the distributions were narrower in the presence of cAMP. Continuous-wave electron paramagnetic resonance studies revealed changes in mobility associated with cAMP binding, indicating less conformational heterogeneity in the cAMP-bound state. From the measured DEER distributions, we constructed a coarse-grained elastic-network structural model of the cAMP-induced conformational transition. We find that binding of cAMP triggers a reorientation of several helices within the CNBD, including the C-helix closest to the cAMP-binding site. These results provide a basis for understanding how the binding of cAMP is coupled to channel opening in HCN and related channels.

Polyvalent Cations Constitute the Voltage Gating Particle in Human Connexin37 Hemichannels

Connexins oligomerize to form intercellular channels that gate in response to voltage and chemical agents such as divalent cations. Historically, these are believed to be two independent processes. Here, data for human connexin37 (hCx37) hemichannels indicate that voltage gating can be explained as block/unblock without the necessity for an independent voltage gate. hCx37 hemichannels closed at negative potentials and opened in a time-dependent fashion at positive potentials. In the absence of polyvalent cations, however, the channels were open at relatively negative potentials, passing current linearly with respect to voltage. Current at negative potentials could be inhibited in a concentration-dependent manner by the addition of polyvalent cations to the bathing solution. Inhibition could be explained as voltage-dependent block of hCx37, with the field acting directly on polyvalent cations, driving them through the pore to an intracellular site. At positive potentials, in the presence of polyvalent cations, the field favored polyvalent efflux from the intracellular blocking site, allowing current flow. The rate of appearance of current depended on the species and valence of the polyvalent cation in the bathing solution. The rate of current decay upon repolarization depended on the concentration of polyvalent cations in the bathing solution, consistent with deactivation by polyvalent block, and was rapid (time constants of tens of milliseconds), implying a high local concentration of polyvalents in or near the channel pore. Sustained depolarization slowed deactivation in a flux-dependent, voltage- and time-independent fashion. The model for hCx37 voltage gating as polyvalent block/unblock can be expanded to account for observations in the literature regarding hCx37 gap junction channel behavior.

The Nucleotide-Binding Sites of SUR1: A Mechanistic Model

ATP-sensitive potassium (KATP) channels comprise four pore-forming Kir6.2 subunits and four modulatory sulfonylurea receptor (SUR) subunits. The latter belong to the ATP-binding cassette family of transporters. KATP channels are inhibited by ATP (or ADP) binding to Kir6.2 and activated by Mg-nucleotide interactions with SUR. This dual regulation enables the KATP channel to couple the metabolic state of a cell to its electrical excitability and is crucial for the KATP channel’s role in regulating insulin secretion, cardiac and neuronal excitability, and vascular tone. Here, we review the regulation of the KATP channel by adenine nucleotides and present an equilibrium allosteric model for nucleotide activation and inhibition. The model can account for many experimental observations in the literature and provides testable predictions for future experiments.

Neonatal Diabetes and the KATP Channel: From Mutation to Therapy

Activating mutations in one of the two subunits of the ATP-sensitive potassium (KATP) channel cause neonatal diabetes (ND). This may be either transient or permanent and, in approximately 20% of patients, is associated with neurodevelopmental delay. In most patients, switching from insulin to oral sulfonylurea therapy improves glycemic control and ameliorates some of the neurological disabilities. Here, we review how KATP channel mutations lead to the varied clinical phenotype, how sulfonylureas exert their therapeutic effects, and why their efficacy varies with individual mutations.

Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels.

KATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’.

Cryo-electron microscopy structures and progress toward a dynamic understanding of KATP channels

Adenosine triphosphate (ATP)–sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic &bgr; cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.

Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding

The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, suggesting that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.

Correction to 'Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels'.

[ Phil. Trans. R. Soc. B 371 , 20150426 (2016; Published 4 July 2016) ([doi:10.1098/rstb.2015.0426][2])][2] The x -axis labels in figure 2 b are incorrect. The corrected figure 2 is given below. ![Figure 2.][2] Figure 2. Effect of rundown on single-channel KATP channel properties. ( a )

New structural insights into the gating movements of CFTR

In Chicago’s Field Museum of Natural History, there is an exhibit highlighting the evolutionary relationships between carnivorans. Glass cases lining two of the four walls of this room display specimens of feliforms, cats and their relatives. The other two walls are devoted to the caniforms, the dogs. In some future Museum of the Natural History of the Macromolecule, one might expect to encounter a similar room in the membrane protein wing. On one side one would find the channels, which form continuous pores in membranes allowing passive movement of ions and solutes down their concentration gradients, and on another side transporters that use the chemical energy of coupled reactions to move solutes against their concentration gradients. A close inspection of this exhibit would reveal that, unlike for the dogs and cats, there is no separation in the display cases between these two classes of membrane proteins. As one strolls past the ATP-binding cassette (ABC) transporters, a superfamily with thousands of members from archaea to mammals, one would arrive at the ABCC family. Members of this family range from ATP hydrolysis–driven transporters (like MRP1), to ion channel modulators (like SUR1 and SUR2), to an ABC protein that is a bona fide ion channel. This molecular dog–cat is known as the cystic fibrosis transmembrane conductance regulator, or CFTR. Because it is a true ion channel, passing tens of millions of Cl− ions per second down their concentration gradient, it is accessible to the tools of electrophysiology. By measuring Cl− currents, CFTR can be studied down to the single-molecule level and with high temporal precision. This is scarcely possible for other ABC proteins. In the April issue of the JGP, Chaves and Gadsby (2015) capitalize on CFTR’s accessibility to electrophysiological assays to probe changes in channel structure that are strongly coupled to the opening and closing of the pore. Their findings confirm an important feature of the CFTR gating cycle and increase our understanding of CFTR as well as those ABC proteins that can’t speak for themselves.

Dynamic measurements for funny channels

Proteins possess the uncanny ability to telegraph signals over long distances. The presence of a ligand, snugly bound to a site on one end of a molecule, can be “felt” by an effector domain several nanometers away. Determining precisely how this task is accomplished is a major challenge for structural biologists. Hypotheses range from the purely dynamic—for example, a binding event affects the conformational possibilities of nearby domains—to the mechanical view whereby a series of structural changes propagates like dominoes from one domain to the next. In PNAS, Saponaro et al. use NMR spectroscopy to examine the first steps in the process that transduces ligand binding into opening of a hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel and present a structural basis for the inhibition of HCN by the accessory protein TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) (1).