Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system

Errors in the measurement of voltage-activated ion channels in cell-attached patch-clamp recordings

Patch-clamp recording techniques have revolutionized understanding of the function and sub-cellular location of ion channels in excitable cells. The cell-attached patch-clamp configuration represents the method of choice to describe the endogenous properties of voltage-activated ion channels in the axonal, somatic and dendritic membrane of neurons, without disturbance of the intracellular milieu. Here, we directly examine the errors associated with cell-attached patch-clamp measurement of ensemble ion channel activity. We find for a number of classes of voltage-activated channels, recorded from the soma and dendrites of neurons in acute brain-slices and isolated cells, that the amplitude and kinetics of ensemble ion channel activity recorded in cell-attached patches is significantly distorted by transmembrane voltage changes generated by the flow of current through the activated ion channels. We outline simple error–correction procedures that allow a more accurate description of the density and properties of voltage-activated channels to be incorporated into computational models of neurons

Persistently modified h-channels after complex febrile seizures convert the seizure-induced enhancement of inhibition to hyperexcitability.

Febrile seizures are the most common type of developmental seizures, affecting up to 5% of children. Experimental complex febrile seizures involving the immature rat hippocampus led to a persistent lowering of seizure threshold despite an upregulation of inhibition. Here we provide a mechanistic resolution to this paradox by showing that, in the hippocampus of rats that had febrile seizures, the long-lasting enhancement of the widely expressed intrinsic membrane conductance Ih converts the potentiated synaptic inhibition to hyperexcitability in a frequency-dependent manner. The altered gain of this molecular inhibition-excitation converter reveals a new mechanism for controlling the balance of excitation-inhibition in the limbic system. In addition, here we show for the first time that h-channels are modified in a human neurological disease paradigm

High-Pass Filtering of Input Signals by the Ih Current in a Non-Spiking Neuron, the Retinal Rod Bipolar Cell

Hyperpolarization–activated cyclic nucleotide–sensitive (HCN) channels mediate the If current in heart and Ih throughout the nervous system. In spiking neurons Ih participates primarily in different forms of rhythmic activity. Little is known, however, about its role in neurons operating with graded potentials as in the retina, where all four channel isoforms are expressed. Intriguing evidence for an involvement of Ih in early visual processing are the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors. Moreover, electroretinographic recordings indicate that these drugs affect temporal processing in the outer retina. Here we analyzed the functional role of HCN channels in rod bipolar cells (RBCs) of the mouse. Perforated–patch recordings in the dark–adapted slice found that RBCs exhibit Ih, and that this is sensitive to the specific blocker ZD7288. RBC input impedance, explored by sinusoidal frequency–modulated current stimuli (0.1–30 Hz), displays band–pass behavior in the range of Ih activation. Theoretical modeling and pharmacological blockade demonstrate that high–pass filtering of input signals by Ih, in combination with low–pass filtering by passive properties, fully accounts for this frequency–tuning. Correcting for the depolarization introduced by shunting through the pipette–membrane seal, leads to predict that in darkness Ih is tonically active in RBCs and quickens their responses to dim light stimuli. Immunohistochemistry targeting candidate subunit isoforms HCN1–2, in combination with markers of RBCs (PKC) and rod–RBC synaptic contacts (bassoon, mGluR6, Kv1.3), suggests that RBCs express HCN2 on the tip of their dendrites. The functional properties conferred by Ih onto RBCs may contribute to shape the retina's light response and explain the visual side effects of HCN inhibitors

The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2–5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity

The JCMT Plane Survey: early results from the l = 30 degree field

We present early results from the JCMT Plane Survey (JPS), which has surveyed the northern inner Galactic plane between longitudes l=7 and l=63 degrees in the 850-{\mu}m continuum with SCUBA-2, as part of the James Clerk Maxwell Telescope Legacy Survey programme. Data from the l=30 degree survey region, which contains the massive star-forming regions W43 and G29.96, are analysed after approximately 40% of the observations had been completed. The pixel-to-pixel noise is found to be 19 mJy/beam, after a smooth over the beam area, and the projected equivalent noise levels in the final survey are expected to be around 10 mJy/beam. An initial extraction of compact sources was performed using the FellWalker method resulting in the detection of 1029 sources above a 5-{\sigma} surface-brightness threshold. The completeness limits in these data are estimated to be around 0.2 Jy/beam (peak flux density) and 0.8 Jy (integrated flux density) and are therefore probably already dominated by source confusion in this relatively crowded section of the survey. The flux densities of extracted compact sources are consistent with those of matching detections in the shallower ATLASGAL survey. We analyse the virial and evolutionary state of the detected clumps in the W43 star-forming complex and find that they appear younger than the Galactic-plane average