Decreased Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Activity in a Rat Model of Absence Epilepsy and the Effect of ZD7288, an Ih Inhibitor, on the Spike-and-Wave Discharges

<b><i>Introduction:</i></b> Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents of <i>Ih</i> and absence epilepsy seizures are associated, but studies reveal differential results. <b><i>Objective:</i></b> In our study, we aimed to investigate the role of the HCN channels on the expression of spike-and-wave discharges (SWDs) using the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. <b><i>Methods:</i></b> HCN isoform levels from isolated brains of both naïve nonepileptic Wistar and GAERS groups were evaluated by enzyme-linked immunosorbent assay. ZD7288, an <i>Ih</i> inhibitor as well as an HCN channel antagonist, was administered intracerebroventricularly to the adult GAERS groups, and to evaluate their SWD activities, electroencephalography was recorded. The effect of ZD7288 on the cumulative total duration and number of SWDs and the mean duration of each SWD complex was evaluated. <b><i>Results:</i></b> The HCN2 levels in the cortex and hippocampus of the GAERS group were lower compared to the naïve nonepileptic Wistar group (<i>p</i> &#x3c; 0.05). ZD7288 increased the number of SWDs at the 20th and 120th min with the highest administered dose of 7 μg (<i>p</i> &#x3c; 0.05). <b><i>Conclusion:</i></b> The <i>Ih</i> inhibitor ZD7288 increased the number of SWDs in a genetic absence epilepsy rat model, although this increase may not be significant due to the inconsistent time-dependent effects. In GAERS, the cortical and hippocampal HCN2 channel levels were significantly lower compared to the control group. Further studies are needed with higher doses of ZD7288 to determine if the effects will increase drastically.

PDE-Mediated Cyclic Nucleotide Compartmentation in Vascular Smooth Muscle Cells: From Basic to a Clinical Perspective

Cardiovascular diseases are important causes of mortality and morbidity worldwide. Vascular smooth muscle cells (SMCs) are major components of blood vessels and are involved in physiologic and pathophysiologic conditions. In healthy vessels, vascular SMCs contribute to vasotone and regulate blood flow by cyclic nucleotide intracellular pathways. However, vascular SMCs lose their contractile phenotype under pathological conditions and alter contractility or signalling mechanisms, including cyclic nucleotide compartmentation. In the present review, we focus on compartmentalized signaling of cyclic nucleotides in vascular smooth muscle. A deeper understanding of these mechanisms clarifies the most relevant axes for the regulation of vascular tone. Furthermore, this allows the detection of possible changes associated with pathological processes, which may be of help for the discovery of novel drugs.

Cyclic Nucleotide (cNMP) Analogues: Past, Present and Future

Cyclic nucleotides are important second messengers involved in cellular events, and analogues of this type of molecules are promising drug candidates. Some cyclic nucleotide analogues have become standard tools for the investigation of biochemical and physiological signal transduction pathways, such as the Rp-diastereomers of adenosine and guanosine 3′,5′-cyclic monophosphorothioate, which are competitive inhibitors of cAMP- and cGMP-dependent protein kinases. Next generation analogues exhibit a higher membrane permeability, increased resistance against degradation, and improved target specificity, or are caged or photoactivatable for fast and/or targeted cellular imaging. Novel specific nucleotide analogues activating or inhibiting cyclic nucleotide-dependent ion channels, EPAC/GEF proteins, and bacterial target molecules have been developed, opening new avenues for basic and applied research. This review provides an overview of the current state of the field, what can be expected in the future and some practical considerations for the use of cyclic nucleotide analogues in biological systems.

Structural and functional approaches to studying cAMP regulation of HCN channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are primarily activated by voltage and further modulated by cAMP. While cAMP binding alone does not open the channel, its presence facilitates the action of voltage, increasing channel open probability. Functional results indicate that the membrane-based voltage sensor domain (VSD) communicates with the cytosolic cyclic nucleotide-binding domain (CNBD), and vice-versa. Yet, a mechanistic explanation on how this could occur in structural terms is still lacking. In this review, we will discuss the recent advancement in understanding the molecular mechanisms connecting the VSD with the CNBD in the tetrameric organization of HCN channels unveiled by the 3D structures of HCN1 and HCN4. Data show that the HCN domain transmits cAMP signal to the VSD by bridging the cytosolic to the membrane domains. Furthermore, a metal ion coordination site connects the C-linker to the S4–S5 linker in HCN4, further facilitating cAMP signal transmission to the VSD in this isoform.

Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. Kinetic modeling suggests that binding of the second ligand is either independent of the first ligand or exhibits up to ~10-fold positive binding cooperativity. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.