Reversible Electrochemotherapy (ECT) as a Treatment Option for Local RCC Recurrence in Solitary Kidney.

A 61-year-old female underwent a right radical nephrectomy and a left nephron sparing surgery in 2014 due to renal cell carcinoma. A renal cell carcinoma local recurrence, 28 mm in size, centrally located in the left kidney was treated using cryoablation in 2016. In November 2018, computed tomography (CT) scan showed three nodules (maximum size 15 × 11 mm) in the left kidney, and CT-guided needle biopsy was performed. For multifocal recurrence and the anatomical site of these three nodules, a simultaneous reversible electrochemotherapy treatment was performed in April 2019. At 6-month CT control, no evidence of residual disease was found. Electrochemotherapy could be used to treat locoregional renal cell carcinoma recurrence when other ablative techniques are not suitable. LEVEL OF EVIDENCE: Level 4, Case Report.

Effects of ospemifene on bone in postmenopausal women.

Ospemifene is a selective estrogen-receptor modulator approved for treating menopause-related moderate to severe dyspareunia and vaginal dryness, symptoms of vulvovaginal atrophy (VVA), in the United States, and for treating menopause-related, symptomatic VVA in women not appropriate for local estrogen therapy in Europe. This review summarizes the effects of ospemifene on bone, including bone biomarker data from a phase 3 vaginal dryness study. Early-phase studies of postmenopausal women showed that ospemifene dose-dependently decreased bone turnover markers versus placebo, similar to raloxifene. A 12-week, phase 3 study of ospemifene 60 mg/day in postmenopausal women showed improvements in all VVA parameters and significantly greater decreases in seven of nine bone biomarkers versus placebo. Lower bone resorption markers with ospemifene were observed regardless of time since menopause (≤5 years or >5 years) or baseline bone mineral density (BMD) (normal [n = 18], osteopenia [n = 164], or osteoporosis [n = 21]). Biomarker studies (n = 565 who took ospemifene) therefore support a potential role for ospemifene in maintaining bone health (and possibly reducing fracture risk) in postmenopausal women taking it for VVA; however, caution is warranted because data are limited to biochemical markers, rather than fracture and BMD. Although studies show that bone turnover predicts BMD and fractures, any hypothesis about a bone-sparing effect of ospemifene needs testing in rigorous, long-term, phase 3 studies monitoring fractures and BMD.

Genitourinary syndrome of menopause in five Asian countries: results from the Pan-Asian REVIVE survey.

OBJECTIVES: The Pan-Asian REVIVE survey aimed to examine women's experiences with genitourinary syndrome of menopause (GSM) and their interactions with health-care professionals (HCPs). METHODS: Self-completed surveys were administered face-to-face to 5992 women (aged 45-75 years) in Indonesia, Malaysia, Singapore, Taiwan, and Thailand. RESULTS: Of 638 postmenopausal women with GSM symptoms, only 35% were aware of the GSM condition, most of whom first heard of GSM through their physician (32%). The most common symptoms were vaginal dryness (57%) and irritation (43%). GSM had the greatest impact on sexual enjoyment (65%) and intimacy (61%). Only 25% had discussed their GSM symptoms with a HCP, and such discussions were mostly patient-initiated (64%) rather than HCP-initiated (24%). Only 21% had been clinically diagnosed with GSM and only 24% had ever used treatment for their symptoms. Three-quarters of those who had used treatment for GSM had discussed their symptoms with a HCP compared to only 9% of those who were treatment-naïve. CONCLUSION: GSM is underdiagnosed and undertreated in Asia. As discussion of GSM with HCPs appears to be a factor influencing women's awareness and treatment status, a more active role by HCPs to facilitate early discussions on GSM and its treatment options is needed.

HOW FUNGI INTERACT WITH NEMATODE TO ACTIVATE THE PLANT DEFENCE RESPONSE TO TOMATO PLANTS.

Management of plant parasitic nematodes with nematode predators, parasites or antagonists is an eco-friendly approach that may avoid the problems arisen by the use of toxic chemicals. Fungi belonging to Trichoderma spp. are well known in literature for their role in control of plant parasitic nematodes. Root-knot nematodes (RKNs), Meloidogyne spp., are obligate parasites that cause the formation of familiar galls on the roots of many cultivated plants. The interaction between the M. incognita motile second stage juveniles (J2s) and the isolate ITEM 908 of Trichoderma harzianum was examined in its effect on the nematode infestation level of susceptible tomato plants. To gain insight into the mechanisms by which ITEM 908 interacts with nematode-infected tomato plants, the expression patterns of the genes PR1 (marker of Salycilic Acid-depending resistance signalling pathway) and JERF3 (marker of the Jasmonic Acid/Ethylene-depending resistance signalling pathway) were detected over time in: i) untreated roots; ii) roots pre-treated with the fungus; iii) roots inoculated with the nematode; iv) pre-treated and inoculated roots. Infestation parameters were checked in untreated plants and plants treated with the fungus to test the effect of the fungus on nematode infestation level and to compare this effect with the expression of the genes PR1 and JERF3, involved in induced resistance.

New strategies in the chemotherapy of leukemia: eradicating cancer stem cells in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by the Philadelphia-positive chromosome deriving from a translocation between chromosomes 22 and 9. The oncogenic product of this aberrant chromosome is the constitutively active tyrosine kinase BCR-ABL that is responsible for leukemic cell growth, proliferation and survival driven by the dysregulation of a large array of signal transduction pathways. Inhibition of BCR-ABL with tyrosine kinase inhibitors proved to be an efficient therapy of CML in the chronic phase. Unfortunately, the impressive success of BCR-ABL inhibitors as front-line therapy in CML has been tempered by problems of disease persistence or relapse arising from different mechanisms, including mutations in the kinase domain of the enzyme BCRABL and mechanisms independent from BCR-ABL activity. Growing evidence has also suggested a pivotal role of persistent leukemic cancer stem cells, characterized by high self-renewal and pluripotency, in CML maintenance and/or relapse. The present review deals with the most recent advances in this challenging field and focuses on the development of new drugs and therapeutic approaches to eradicate the subtle and dangerous leukemic stem cells responsible for maintaining and sustaining tumor growth.

Post-natal cardiomyocytes can generate iPS cells with an enhanced capacity toward cardiomyogenic re-differentation.

Adult mammalian cells can be reprogrammed to a pluripotent state by forcing the expression of a few embryonic transcription factors. The resulting induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers. It is well known that post-natal cardiomyocytes (CMs) lack the capacity to proliferate. Here, we report that neonatal CMs can be reprogrammed to generate iPS cells that express embryonic-specific markers and feature gene-expression profiles similar to those of mouse embryonic stem (mES) cell and cardiac fibroblast (CF)-derived iPS cell populations. CM-derived iPS cells are able to generate chimeric mice and, moreover, re-differentiate toward CMs more efficiently then either CF-derived iPS cells or mES cells. The increased differentiation capacity is possibly related to CM-derived iPS cells retaining an epigenetic memory of the phenotype of their founder cell. CM-derived iPS cells may thus lead to new information on differentiation processes underlying cardiac differentiation and proliferation.

Caffeine-induced Ca(2+) signaling as an index of cardiac progenitor cells differentiation.

Cardiac progenitor cells (CPCs), migrating from heart tissue, in culture aggregate to form cardiospheres (CSs) in which replication and cardiogenic differentiation occur. However, the frequency of functional differentiation in CSs and the role of cell clustering in supporting it remain to be established. The aim of our study is to quantify differentiation of a muscle-type Ca(2+) release mechanism in CS-derived cells, correlate it with cardiac differentiation markers and test its dependency on CS formation. CPCs migrating from murine cardiac explants were studied prior and after CSs formation (Pre-CS and Post-CS). Inducibility of RyR- and IP3-R-mediated Ca(2+) transients in individual cells was tested by exposure to caffeine and ATP, respectively; expression of cardiac and non-cardiac lineage markers was assessed. Caffeine responsiveness was negligible in Pre-CS cells and increased by 7.5 fold in Post-CS cells (3.6 vs. 26.9%; p < 0.05), and was closely correlated with activation of the cardiac TnI gene promoter. ATP-induced responses, frequent in Pre-CS (86%), were slightly increased in Post-CS cells (94%; p < 0.05). Expression of cardiac-specific Ca(2+)-handling proteins (Cav1.2, NCX1, RyR2, SERCA2a) was either limited to the Post-CS stage, or markedly enhanced. CS beating was infrequent, but its pharmacology was compatible with cardiac excitation-contraction coupling. Expression of non-cardiac lineage was low in general, and similar between Pre- and Post-CS cells. Culture conditions inhibiting CSs formation prevented the increase in caffeine responders. In conclusion, clustering in CSs leads to the induction of a muscle-specific functional response in about 30% of CPCs; this is accompanied by development of a cardiac-specific expression pattern.

Toxicity assessment of metabolites of fungal biocontrol agents using two different (Artemia salina and Daphnia magna) invertebrate bioassays.

Fungal biocontrol agents (BCAs) have been marketed for control of crop pests, weeds, and diseases. However, BCAs may produce toxic metabolites, whose presence in the formulated products, in the crops and in the environment should be considered along with the associated risk. Two invertebrate models, viz. Artemia salina and Daphnia magna were used to assess the acute toxicity of seven BCA metabolites, characterized by different chemical nature and mode of action, namely alamethicin (ALA), paracelsin (PCS), antiamoebin (AAM), gliotoxin (GTX), destruxin A (DA), oosporein (OOS), and elsinochrome A (EA). The two invertebrates were very sensitive to all the metabolites examined, except OOS. The LC50s after 24 and 36 h exposures showed the following toxicity ranks: A. salina, DA > ALA > EA > GTX > AAM > PCS (LC50s ranging from 9.78 to 40.84 microg/ml at 24 h and from 2.92 to 18.56 microg/ml at 36 h); D. magna, DA > GTX = EA > ALA > PCS > AAM (LC50s ranging from 0.20 to 24.41 microg/ml at 24h and from 0.16 to 11.98 microg/ml at 36 h). LC50 of OOS to D. magna increased dramatically in 36 h exposure, compared to 24 h exposures (5.84 and 68.40 microg/ml, respectively). A. salina and D. magna proved to be suitable models for rapid and inexpensive screening of toxicity of BCAs at an early stage of product development.

Rate dependency of beta-adrenergic modulation of repolarizing currents in the guinea-pig ventricle.

Beta-adrenergic stimulation modulates ventricular currents and sinus cycle length (CL). We investigated how changes in CL affect the current induced by isoprenaline (Iso) during the action potential (AP) of guinea-pig ventricular myocytes. Action-potential clamp was applied at CLs of 250 and 1000 ms to measure: (1) the net current induced by 0.1 microm Iso (I(Iso)); (2) the L-type Ca2+ current I(CaL) and slow delayed rectifier current I(Ks) components of I(Iso) (I(IsoCa) and I(IsoK)), identified as the Iso-induced current sensitive to nifedipine and HMR1556, respectively; and (3) I(Iso) persisting after inhibition of both I(Ca) and I(Ks) (I(isoR)). The pause dependency of I(Ks) and its modulation were evaluated in voltage-clamp experiments. The rate dependency of the duration of the action potential at 90% repolarization (APD90) and its modulation by isoprenaline were tested in current-clamp experiments. At a CL of 250 ms I(Iso) was inward during initial repolarization and reversed at 59% of APD90. At a CL of 1000 ms I(Iso) became mostly inward in all cells. Switching to shorter CL did not change I(IsoCa) and I(IsoK) amplitudes, but moved their peak amplitudes to earlier repolarization; I(IsoR) was independent of CL. Acceleration of I(IsoK) at shorter CL was based on faster pause dependency of I(Ks) activation rate. The 'restitution' of activation rates was modulated by isoprenaline. The APD90-CL relation was rotated anticlockwise by isoprenaline and crossed the control curve at a CL of 150 ms (400 beats min(-1)). We conclude that: (1) isoprenaline induced markedly different current profiles according to pacing rate, involving CL-dependent I(Ca) and I(Ks) modulation; (2) the effect of isoprenaline on APD90 was CL dependent, and negligible during tachycardia; and (3) during sympathetic activation, repolarization stability may involve matched modulation of sinus rate and repolarizing currents.

Coumarins derivatives as dual inhibitors of acetylcholinesterase and monoamine oxidase.

A set of 17 coumarin and 2 chromone derivatives with known inhibitory activity toward monoamine oxidase (MAO) A and B were tested as acetylcholinesterase (AChE) inhibitors. All compounds inhibited AChE with values in the micromolar range (3-100 microM). A kinetic study showed that most compounds acted as noncompetitive AChE inhibitors. This finding may be of interest in the context of Alzheimer's disease because recent observations suggest that MAO and AChE inhibition might decrease beta-amyloid deposition.

C terminus-mediated control of voltage and cAMP gating of hyperpolarization-activated cyclic nucleotide-gated channels.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) family of "pacemaker" channels includes 4 isoforms, the kinetics and cAMP-induced modulation of which differ quantitatively. Because HCN isoforms are highly homologous in the central region, but diverge more substantially in the N and C termini, we asked whether these latter regions could contribute to the determination of channel properties. To this aim, we analyzed activation/deactivation kinetics and the response to cAMP of heterologously expressed isoforms mHCN1 and rbHCN4 and verified that mHCN1 has much faster kinetics and lower cAMP sensitivity than rbHCN4. We then constructed rbHCN4 chimeras by replacing either the N or the C terminus, or both, with the analogous domains from mHCN1. We found that: 1) replacement of the N terminus (chimera N1-4) did not substantially modify either the kinetics or cAMP dependence of wild-type channels; 2) replacement of the C terminus, on the contrary, resulted in a chimeric channel (4-C1), the kinetics of which were strongly accelerated compared with rbHCN4, and that was fully insensitive to cAMP; 3) replacement of both N and C termini led to the same results as replacement of the C terminus alone. These results indicate that the C terminus of rbHCN4 contributes to the regulation of voltage- and cAMP-dependent channel gating, possibly through interaction with other intracellular regions not belonging to the N terminus.

Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current I(f).

The pacemaker current I(f) of the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those of I(f), suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.

Integrated allosteric model of voltage gating of HCN channels.

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.

High affinity central benzodiazepine receptor ligands. Part 2: quantitative structure-activity relationships and comparative molecular field analysis of pyrazolo[4,3-c]quinolin-3-ones.

A large series of 2-aryl(heteroaryl)-2,5-dihydropyrazolo[4,3-c]quinolin-3(3H)-ones (PQ, 106 compounds), carrying appropriate substituents at the quinoline and N2-phenyl rings, were designed, prepared and tested as central benzodiazepine receptor ligands. Compounds with an affinity significantly higher than the parent compound CGS-8216 were obtained, the most active ligand showing a pIC50 = 10.35. Hansch and comparative molecular field analyses gave coherent results suggesting the main structural requirements of high receptor binding affinity. The possible formation of a three-centred hydrogen bond (HB) at the HB donor site H2, as a key interaction for high receptor binding affinity, was assessed by the calculation and comparison of the molecular electrostatic potentials of a series of selected ligands.

Neuronal nicotinic receptor agonists: a multi-approach development of the pharmacophore.

Based on the results obtained with different automated computational approaches as applied to the study of eleven high-affinity agonists of the neuronal nicotine acetylcholine receptor (nAChR), belonging to different chemical classes, new relevant features were detected which complement the existing pharmacophores. Convergent results from DISCO (Distance Comparison), QXP (Quick Explore), Catalyst/HipHop, and MIPSIM (Molecular Interaction Potential Similarity) allowed us to identify and locate, in a well defined spatial arrangement, three geometrically independent key structural features: (i) a positively charged nitrogen atom for ionic or hydrogen bond interactions, (ii) a lone pair of the pyridine nitrogen or a specific lone pair of a carbonyl oxygen, as a hydrogen bond acceptor, and (iii) a centre of a hydrophobic area generally occupied by aliphatic cycles. The pharmacophore presented herein, along with predictive 2D and 3D QSAR models recently developed in our group, could represent valuable computational tools for the design of new nAChR agonists having therapeutical potential.

Inhibition of monoamine oxidases by functionalized coumarin derivatives: biological activities, QSARs, and 3D-QSARs.

A large series of coumarin derivatives (71 compounds) were tested for their monoamine oxidase A and B (MAO-A and MAO-B) inhibitory activity. Most of the compounds acted preferentially on MAO-B with IC(50) values in the micromolar to low-nanomolar range; high inhibitory activities toward MAO-A were also measured for sulfonic acid esters. The most active compound was 7-[(3, 4-difluorobenzyl)oxy]-3,4-dimethylcoumarin, with an IC(50) value toward MAO-B of 1.14 nM. A QSAR study of 7-X-benzyloxy meta-substituted 3,4-dimethylcoumarin derivatives acting on MAO-B yielded good statistical results (q(2)() = 0.72, r(2)() = 0.86), revealing the importance of lipophilic interactions in modulating the inhibition and excluding any dependence on electronic properties. CoMFA was performed on two data sets of MAO-A and MAO-B inhibitors. The GOLPE procedure, with variable selection criteria, was applied to improve the predictivity of the models and to facilitate the graphical interpretation of results.

Biological characterization of fusapyrone and deoxyfusapyrone, two bioactive secondary metabolites of Fusarium semitectum.

Fusapyrone (1) and deoxyfusapyrone (2), two alpha-pyrones originally isolated from rice cultures of Fusarium semitectum, were tested in several biological assays. Compounds 1 and 2 showed considerable antifungal activity against several plant pathogenic and/or mycotoxigenic filamentous fungi, although they were inactive toward yeasts isolated from plants and the Gram-positive bacterium Bacillus megaterium in disk diffusion assays. Compound 1 was consistently more active than 2. Among the tested fungi, Fusarium species were the least sensitive to the two pyrones, while Alternaria alternata, Ascochyta rabiei, Aspergillusflavus, Botrytis cinerea, Cladosporium cucumerinum, Phoma tracheiphila, and Penicillium verrucosum were the most sensitive. Compounds 1 and 2 also showed good inhibitory activity toward agents of human mycoses. Aspergilli were the most sensitive, while some species-specific variability was found among the Candida spp. In an Artemia salina larvae bioassay, 1 was not toxic at the highest concentration tested (500 microM), whereas the LC(50) of 2 was 37.1 microM (21.8 microg/mL). Neither 1 nor 2 was phytotoxic in a panel of assays that monitored plant-cell toxicity, as well as wilt-, chlorosis-, and necrosis-inducing activity. Moreover, 2 stimulated the root elongation of tomato seedlings at doses of 10 and 100 microM. In consideration of the biological activities evidenced in this study, 1 and 2 appear to be potential candidates for biotechnological applications, as well as good models for studies on mechanism(s) of action and structure-activity relationships.

Ionization behaviour and tautomerism-dependent lipophilicity of pyridine-2(1H)-one cardiotonic agents.

The acid-base properties of pyridine-2(1H)-one derivatives, analogues of the cardiotonic agent milrinone, were studied by capillary zone electrophoresis (CZE). Electrophoretic mobility and pH data were fitted to equilibrium expressions and apparent dissociation constants (pKa) calculated by non-linear regression. Compared with the ultraviolet (UV) spectrophotometric method and potentiometric titrations, the CZE technique showed advantages, such as rapidity and applicability to compounds that are sparingly soluble in water. Based on the pKa values, intramolecular electronic interactions were assessed. The lipophilicity of a number of derivatives was also examined, by determining their n-octanol/water distribution coefficients over a wide pH range, and found to be significantly affected by 2-pyridone/2-hydroxypyridine tautomerism. As revealed by a comparison between experimental and calculated log P values, electron withdrawing substituents, especially at the C(6) position of 2-pyridone, favour the less polar hydroxypyridine tautomers both in water and octanol. Our results indicate that the positive inotropism of milrinone-related compounds could be explained taking ionization and tautomerism into account.

Effects of dronedarone on acetylcholine-activated current in rabbit SAN cells.

1. The effect of the antiarrhythmic drug dronedarone on the Acetylcholine-activated K(+) current (I(K(ACh))) was investigated in single cells isolated from sinoatrial node (SAN) tissue of rabbit hearts. 2. Externally perfused dronedarone (0.001 - 1 microM) caused a potent, voltage independent block of I(K(ACh)). Fitting of the dose response curve of I(K(ACh)) block yielded an IC(50) value of 63 nM, a value over one order of magnitude lower than those reported for dronedarone block of other cardiac currents. 3. I(K(ACh)) block was not due to an inhibitory action of dronedarone on the muscarinic M2 receptor activation, since the drug was effective on I(K(ACh)) constitutively activated by intracellular perfusion with GTP-gammaS. 4. External cell perfusion with dronedarone inhibited the activity of I(K(ACh)) channels recorded from cell-attached patches by reducing the channel open probability (from 0.56 to 0.11) without modification of the single-channel conductance. 5. These data suggest that dronedarone blocks I(K(ACh)) channels either by disrupting the G-protein-mediated activation or by a direct inhibitory interaction with the channel protein.

Kinetic and ionic properties of the human HCN2 pacemaker channel.

Human cDNA coding for the hyperpolarization-activated "pacemaker" channel HCN2 was expressed in Phoenix cells and yielded an inward current (IhHCN2) activated on hyperpolarization. The average IhHCN2 was half-activated at -83.1 mV and its kinetics could be described by second-order Hodgkin-Huxley gating. The time constant curve was bell-shaped and peaked at -82.2 mV. With 115 mM external Na+ and 30 mM external K+, IhHCN2 reversed at -17.1 mV, and had a mean conductance of 5.6 nS. Reducing the external K+ or Na+ concentration led to a concentration-dependent reduction of the IhHCN2 conductance and to a hyperpolarizing shift of reversal potential. External Cs+ ions (5 mM) blocked IhHCN2 in a voltage-dependent way according to a Woodhull-type block model, at an electrical distance of 0.66 from the external membrane surface, and with a dissociation constant of 15 mM at 0 mV. Increasing cytoplasmic cAMP using forskolin increased IhHCN2 by shifting the current activation curve to more positive voltages (11.7 mV). Exposure of the intracellular side of inside-out macro-patches to cAMP led to a depolarizing shift of the channel open probability curve (15.2 mV with 10 microM cAMP). These results indicate that although hHCN2 channels share several properties with native cardiac f-channels, differences also exist in permeability and block properties, suggesting that native channels may not be composed simply of homomeric constructs.