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 Arg237 and Gly239 residues on the S2 of the VSD that form direct interactions with Ile135 on the HCND. Disrupting these interactions abolished HCN2 currents. We also identified three residues on the C-linker-CNBD (Glu478, Gln482, and His559) that form direct interactions with residues Arg154 and Ser158 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.

Increased amplitude of inward rectifier K+ currents with advanced age in smooth muscle cells of murine superior epigastric arteries.

Inward rectifier K+ channels (KIR) may contribute to skeletal muscle blood flow regulation and adapt to advanced age. Using mouse abdominal wall superior epigastric arteries (SEAs) from either young (3-6 mo) or old (24-26 mo) male C57BL/6 mice, we investigated whether SEA smooth muscle cells (SMCs) express functional KIR channels and how aging may affect KIR function. Freshly dissected SEAs were either enzymatically dissociated to isolate SMCs for electrophysiological recording (perforated patch) and mRNA expression or used intact for pressure myography. With 5 mM extracellular K+ concentration ([K+]o), exposure of SMCs to the KIR blocker Ba2+ (100 µM) had no significant effect (P > 0.05) on whole cell currents elicited by membrane potentials spanning -120 to -30 mV. Raising [K+]o to 15 mM activated Ba2+-sensitive KIR currents between -120 and -30 mV, which were greater in SMCs from old mice than in SMCs from young mice (P < 0.05). Pressure myography of SEAs revealed that while aging decreased maximum vessel diameter by ~8% (P < 0.05), it had no significant effect on resting diameter, myogenic tone, dilation to 15 mM [K+]o, Ba2+-induced constriction in 5 mM [K+]o, or constriction induced by 15 mM [K+]o in the presence of Ba2+ (P > 0.05). Quantitative RT-PCR revealed SMC expression of KIR2.1 and KIR2.2 mRNA that was not affected by age. Barium-induced constriction of SEAs from young and old mice suggests an integral role for KIR in regulating resting membrane potential and vasomotor tone. Increased functional expression of KIR channels during advanced age may compensate for other age-related changes in SEA function.NEW & NOTEWORTHY Ion channels are integral to blood flow regulation. We found greater functional expression of inward rectifying K+ channels in smooth muscle cells of resistance arteries of mouse skeletal muscle with advanced age. This adaptation to aging may contribute to the maintenance of vasomotor tone and blood flow regulation during exercise.

Urothelial purine release during filling of murine and primate bladders.

During urinary bladder filling the bladder urothelium releases chemical mediators that in turn transmit information to the nervous and muscular systems to regulate sensory sensation and detrusor muscle activity. Defects in release of urothelial mediators may cause bladder dysfunctions that are characterized with aberrant bladder sensation during bladder filling. Previous studies have demonstrated release of ATP from the bladder urothelium during bladder filling, and ATP remains the most studied purine mediator that is released from the urothelium. However, the micturition cycle is likely regulated by multiple purine mediators, since various purine receptors are found present in many cell types in the bladder wall, including urothelial cells, afferent nerves, interstitial cells in lamina propria, and detrusor smooth muscle cells. Information about the release of other biologically active purines during bladder filling is still lacking. Decentralized bladders from C57BL/6 mice and Cynomolgus monkeys (Macaca fascicularis) were filled with physiological solution at different rates. Intraluminal fluid was analyzed by high-performance liquid chromatography with fluorescence detection for simultaneous evaluation of ATP, ADP, AMP, adenosine, nicotinamide adenine dinucleotide (NAD+), ADP-ribose, and cADP-ribose content. We also measured ex vivo bladder filling pressures and performed cystometry in conscious unrestrained mice at different filling rates. ATP, ADP, AMP, NAD+, ADPR, cADPR, and adenosine were detected released intravesically at different ratios during bladder filling. Purine release increased with increased volumes and rates of filling. Our results support the concept that multiple urothelium-derived purines likely contribute to the complex regulation of bladder sensation during bladder filling.

Aging increases capacitance and spontaneous transient outward current amplitude of smooth muscle cells from murine superior epigastric arteries.

Large conductance Ca(2+)-activated K(+) channels (BKCa) contribute to negative feedback regulation of smooth muscle cell (SMC) tone. However, the effects of aging on BKCa function are unclear. We tested the hypothesis that aging alters SMC BKCa function in superior epigastric arteries (SEAs) by using perforated patch recording of enzymatically isolated SMCs from 3- to 4-mo-old male C57BL/6 mice (Young) and 24- to 26-mo-old male C57BL/6 mice (Old). SMC capacitance from Young (15.7 ± 0.4 pF; n = 110) was less than Old (17.9 ± 0.5 pF; n = 104) (P < 0.05). SMCs displayed spontaneous transient outward currents (STOCs) at membrane potentials more positive than -30 mV; depolarization increased STOC amplitude and frequency (P < 0.05; n = 19-24). STOC frequency in Young (2.2 ± 0.6 Hz) was less than Old (4.2 ± 0.7 Hz) at -10 mV (P < 0.05, n = 27-30), with no difference in amplitude (1.0 ± 0.1 vs. 0.9 ± 0.1 pA/pF, respectively). At +30 mV, STOC amplitude in Young (3.2 ± 0.3 pA/pF) was less than Old (5.0 ± 0.5 pA/pF; P < 0.05, n = 61-67) with no difference in frequency (3.9 ± 0.4 vs. 3.2 ± 0.3 Hz, respectively). BKCa blockers (1 µM paxilline, 100 nM iberiotoxin, 1 mM tetraethylammonium) or a ryanodine receptor antagonist (100 µM tetracaine) inhibited STOCs (n ≥ 6; P < 0.05 each). Western blots revealed increased expression of BKCa α-subunit protein in Old. Pressure myography revealed no effect of age on SEA maximal diameter, myogenic tone, or paxilline-induced constriction (n = 10-12; P > 0.05). Enhanced functional expression of SMC BKCa-dependent STOCs in Old may represent an adaptation of resistance arteries to maintain functional integrity.

Constitutive and evoked release of ATP in adult mouse olfactory epithelium.

In adult olfactory epithelium (OE), ATP plays a role in constant cell turnover and post-injury neuroregeneration. We previously demonstrated that constitutive and ATP-evoked ATP release are present in neonatal mouse OE and underlie continuous cell turn-over and post-injury neuroregeneration, and that activation of purinergic P2X7 receptors is involved in the evoked release. We hypothesized that both releases are present in adult mouse OE. To study the putative contribution of olfactory sensory neurons to ATP release, we used olfactory sensory neuronal-like OP6 cells derived from the embryonic olfactory placode cells. Calcium imaging showed that OP6 cells and primary adult OE cell cultures express functional purinergic receptors. We monitored ATP release from OP6 cells and whole adult OE turbinates using HEK cells as biosensors and luciferin-luciferase assays. Constitutive ATP release occurs in OP6 cells and whole adult mouse OE turbinates, and P2X7 receptors mediated evoked ATP release occurs only in turbinates. The mechanisms of ATP release described in the present study might underlie the constant cell turn-over and post-injury neuroregeneration present in adult OE and thus, further studies of these mechanisms are warranted as it will improve our knowledge of OE tissue homeostasis and post-injury regeneration.

Neuroimmune interplay in kidney health and disease: Role of renal nerves.

Renal nerves and their role in physiology and disease have been a topic of increasing interest in the past few decades. Renal inflammation contributes to many cardiorenal disease conditions, including hypertension, chronic kidney disease, and polycystic kidney disease. Much is known about the role of renal sympathetic nerves in physiology - they contribute to the regulation of sodium reabsorption, renin release, and renal vascular resistance. In contrast, far less is known about afferent, or "sensory," renal nerves, which convey signals from the kidney to the brain. While much remains unknown about these nerves in the context of normal physiology, even less is known about their contribution to disease states. Furthermore, it has become apparent that the crosstalk between renal nerves and the immune system may augment or modulate disease. Research from other fields, especially pain research, has provided critical insight into neuroimmune crosstalk. Sympathetic renal nerve activity may increase immune cell recruitment, but far less work has been done investigating the interplay between afferent renal nerves and the immune system. Evidence from other fields suggests that inflammation may augment afferent renal nerve activity. Furthermore, these nerves may exacerbate renal inflammation through the release of afferent-specific neurotransmitters.

Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium.

BACKGROUND: ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s) by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. RESULTS: Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αßMeATP and Bz-ATP) and a G protein-coupled P2Y receptor agonist (UTP). Calcium imaging of P2X2-transfected HEK293 "biosensor" cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. CONCLUSIONS: The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP release following injury could lead to progenitor cell proliferation, differentiation and regeneration. Thus, understanding mechanisms of ATP release is of paramount importance to improve our knowledge about tissue homeostasis and post-injury neuroregeneration. It will lead to development of treatments to restore loss of smell and, when transposed to the central nervous system, improve recovery following central nervous system injury.

Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.

A delayed ATP-elicited K+ current in freshly isolated smooth muscle cells from mouse aorta.

Adenosine 5'-triphosphate (ATP) activated two sequential responses in freshly isolated mouse aortic smooth muscle cells. In the first phase, ATP activated Ca(2+)-dependent K(+) or Cl(-) currents and the second phase was the activation of a delayed outward current with a reversal potential of -75.9 +/- 1.4 mV. A high concentration of extracellular K(+) (130 mM) shifted the reversal potential of the delayed ATP-elicited current to -3.5 +/- 1.3 mV. The known K(+)-channel blockers, iberiotoxin, charybdotoxin, glibenclamide, apamin, 4-aminopyridine, Ba(2+) and tetraethylammonium chloride all failed to inhibit the delayed ATP-elicited K(+) current. Removal of ATP did not decrease the amplitude of the ATP-elicited current back to the control values. The simultaneous recording of cytosolic free Ca(2+) and membrane currents revealed that the first phase of the ATP-elicited response is associated with an increase in intracellular Ca(2+), while the second delayed phase develops after the return of cytosolic free Ca(2+) to control levels.ATP did not activate Ca(2+)-dependent K(+) currents, but did elicit Ca(2+)-independent K(+) currents, in cells dialyzed with ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The delay of activation of Ca(2+)-independent currents decreased from 10.5 + 3.4 to 1.27 +/- 0.33 min in the cells dialyzed with 2 mM EGTA. Adenosine alone failed to elicit a Ca(2+)-independent K(+) current but simultaneous application of ATP and adenosine activated the delayed K(+) current. Intracellular dialysis of cells with guanosine 5'-O-(2-thiodiphosphate) transformed the Ca(2+)-independent ATP-elicited response from a sustained to a transient one. A phospholipase C inhibitor, U73122 (1 microM), was shown to abolish the delayed ATP-elicited response. These results indicate that the second phase of the ATP-elicited response was a delayed Ca(2+)-independent K(+) current activated by exogenous ATP. This phase might represent a new vasoregulatory pathway in vascular smooth muscle cells.

Protein kinase A and C regulate leak potassium currents in freshly isolated vascular myocytes from the aorta.

We tested the hypothesis that protein kinase A (PKA) inhibits K2P currents activated by protein kinase C (PKC) in freshly isolated aortic myocytes. PDBu, the PKC agonist, applied extracellularly, increased the amplitude of the K2P currents in the presence of the "cocktail" of K(+) channel blockers. Gö 6976 significantly reduced the increase of the K2P currents by PDBu suggesting the involvement of either α or ß isoenzymes of PKC. We found that forskolin, or membrane permeable cAMP, did not inhibit K2P currents activated by the PKC. However, when PKA agonists were added prior to PDBu, they produced a strong decrease in the K2P current amplitudes activated by PKC. Inhibition of PDBu-elicited K2P currents by cAMP agonists was not prevented by the treatment of vascular smooth muscle cells with PKA antagonists (H-89 and Rp-cAMPs). Zn(2+) and Hg(2+) inhibited K2P currents in one population of cells, produced biphasic responses in another population, and increased the amplitude of the PDBu-elicited K(+) currents in a third population of myocytes, suggesting expression of several K2P channel types. We found that cAMP agonists inhibited biphasic responses and increase of amplitude of the PDBu-elicited K2P currents produced by Zn(2+) and Hg(2). 6-Bnz-cAMp produced a significantly altered pH sensitivity of PDBu-elicited K2P-currents, suggesting the inhibition of alkaline-activated K2P-currents. These results indicate that 6-Bnz-cAMP and other cAMP analogs may inhibit K2P currents through a PKA-independent mechanism. cAMP analogs may interact with unidentified proteins involved in K2P channel regulation. This novel cellular mechanism could provide insights into the interplay between PKC and PKA pathways that regulate vascular tone.

An IP3R3- and NPY-expressing microvillous cell mediates tissue homeostasis and regeneration in the mouse olfactory epithelium.

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3(+/-), and IP3R3(-/-) mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3(-/-) mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3(-/-) mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3(-/-) mice. The reductions in both NPY release and number of progenitor cells in IP3R3(-/-) mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.

Intracellular cAMP: the "switch" that triggers on "spontaneous transient outward currents" generation in freshly isolated myocytes from thoracic aorta.

Spontaneous transient outward currents (STOCs) have been reported in resistance and small arteries but have not yet been found in thoracic aorta. Do thoracic aorta myocytes possess cellular machinery that generates STOCs? It was found that the majority of aortic myocytes do not generate STOCs. STOCs were generated in 8.7% of freshly isolated aortic myocytes. Myocytes that did not generate STOCs we have called "silent" myocytes and myocytes with STOCs have been called "active." STOCs recorded in active myocytes were voltage dependent and were inhibited by ryanodine, caffeine, and charybdotoxin. Forskolin was reported to increase STOCs frequency in myocytes isolated from resistance arteries. Forskolin (10 microM) triggered STOCs generation in 35.1% of silent aortic myocytes. In 36.8% percent of silent myocytes, forskolin did not trigger STOCs but increased the amplitude of charybdotoxin-sensitive outward net current to 136.1 +/- 8.5% at 0 mV. Membrane-permeable 8BrcAMP triggered STOCs generation in 38.7% of silent myocytes. Forskolin- or 8BrcAMP-triggered STOCs were inhibited by charybdotoxin. 8BrcAMP also increased open probability of BK(Ca) channels in BAPTA-AM-pretreated cells. Our data demonstrate that, in contrast to resistance arteries, STOCs are present just in the minority of myocytes in the thoracic aorta. However, cellular machinery that generates STOCs can be "switched" on by cAMP. Such an inactive cellular mechanism could modulate the contractility of the thoracic aorta in response to physiological demand.