Genetically engineered cardiac pacemaker: stem cells transfected with HCN2 gene and myocytes - a model

Artificial biological pacemakers were developed and tested in canine ventricles. Next steps will require obtaining oscillations sensitive to external regulations, and robust with respect to long term drifts of expression levels of pacemaker currents and gap junctions. We introduce mathematical models intended to be used in parallel with the experiments. The models describe human mesenchymal stem cells ({\it hMSC}) transfected with HCN2 genes and connected to myocytes. They are intended to mimic experiments with oscillation induction in a cell pair, in cell culture and in the cardiac tissue. We give examples of oscillations in a cell pair, in a 1 dim cell culture, and oscillation dependence on number of pacemaker channels per cell and number of gap junctions. The models permit to mimic experiments with levels of gene expressions not achieved yet, and to predict if the work to achieve this levels will significantly increase the quality of oscillations. This give arguments for selecting the directions of the experimental work

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

Impaired Dendritic Expression and Plasticity Of H-Channels in the fmr1(-/Y) Mouse Model of Fragile X Syndrome

Despite extensive research into both synaptic and morphological changes, surprisingly little is known about dendritic function in fragile X syndrome (FXS). We found that the dendritic input resistance of CA1 neurons was significantly lower in fmr1(-/y) versus wild-type mice. Consistent with elevated dendritic I-h, voltage sag, rebound, and resonance frequency were significantly higher and temporal summation was lower in the dendrites of fmr1(-/y) mice. Dendritic expression of the h-channel subunit HCN1, but not HCN2, was higher in the CA1 region of fmr1(-/y) mice. Interestingly, whereas mGluR-mediated persistent decreases in Ih occurred in both wildtype and fmr1(-/y) mice, persistent increases in Ih that occurred after LTP induction in wild-type mice were absent in fmr1(-/y) mice. Thus, chronic upregulation of dendritic Ih in conjunction with impairment of homeostatic h-channel plasticity represents a dendritic channelopathy in this model of mental retardation and may provide a mechanism for the cognitive impairment associated with FXS.FRAXAUniversity of Texas Austin Undergraduate Research FellowshipNational Institutes of Health Grant MH048432Center for Learning and Memor

Elementary Functional Properties of Single HCN2 Channels

AbstractHyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are tetramers that evoke rhythmic electrical activity in specialized neurons and cardiac cells. These channels are activated by hyperpolarizing voltage, and the second messenger cAMP can further enhance the activation. Despite the physiological importance of HCN channels, their elementary functional properties are still unclear. In this study, we expressed homotetrameric HCN2 channels in Xenopus oocytes and performed single-channel experiments in patches containing either one or multiple channels. We show that the single-channel conductance is as low as 1.67 pS and that channel activation is a one-step process. We also observed that the time between the hyperpolarizing stimulus and the first channel opening, the first latency, determines the activation process alone. Notably, at maximum hyperpolarization, saturating cAMP drives the channel to open for unusually long periods. In particular, at maximum activation by hyperpolarization and saturating cAMP, the open probability approaches unity. In contrast to other reports, no evidence of interchannel cooperativity was observed. In conclusion, single HCN2 channels operate only with an exceptionally low conductance, and both activating stimuli, voltage and cAMP, exclusively control the open probability

Activity-Dependent Regulation of HCN Pacemaker Channels by Cyclic AMP Signaling through Dynamic Allosteric Coupling

AbstractSignal transduction in neurons is a dynamic process, generally thought to be driven by transient changes in the concentration of second messengers. Here we describe a novel regulatory mechanism in which the dynamics of signaling through cyclic AMP are mediated by activity-dependent changes in the affinity of the hyperpolarization-activated, cation nonselective (HCN) channels for cAMP, rather than by changes in cAMP concentration. Due to the allosteric coupling of channel opening and ligand binding, changes in cellular electrical activity that alter the opening of the HCN channels modify the binding of static, basal levels of cAMP. These changes in ligand binding produce long-lasting changes in channel function which can contribute to the regulation of rhythmic firing patterns

The HCN domain is required for HCN channel cell-surface expression and couples voltage- and cAMP-dependent gating mechanisms

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity, and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD) and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane segment via the C-linker. Previous functional analysis of HCN channels has suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of this coupling remain unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed the HCN domain (HCND), forms a direct structural link between the VSD and C-linker/CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified R237 and G239 residues on the S2 of the VSD that form direct interactions with I135 on the HCND. Disrupting these interactions abolished HCN2 currents. We also identified three residues on the C-linker/CNBD (E478, Q382 and H559) that form direct interactions with residues R154 and S158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the cell-surface expression of HCN channels, and provides a functional link between voltage- and cAMP-dependent mechanisms of HCN channel gating