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Proper β-adrenergic signaling is indispensable for modulating heart frequency. Studies on extremely-low-frequency pulsed electromagnetic field (ELF-PEMF) effects in the heart beat function are contradictory and no definitive conclusions were obtained so far. To investigate the interplay between ELF-PEMF exposure and β-adrenergic signaling, cultures of primary murine neonatal cardiomyocytes and of sinoatrial node were exposed to ELF-PEMF and short and long-term effects were evaluated. The ELF-PEMF generated a variable magnetic induction field of 0-6mT at a frequency of 75Hz. Exposure to 3mT ELF-PEMF induced a decrease of contraction rate, Ca(2+) transients, contraction force, and energy consumption both under basal conditions and after β-adrenergic stimulation in neonatal cardiomyocytes. ELF-PEMF exposure inhibited β-adrenergic response in sinoatrial node (SAN) region. ELF-PEMF specifically modulated β2 adrenergic receptor response and the exposure did not modify the increase of contraction rate after adenylate cyclase stimulation by forskolin. In HEK293T cells transfected with β1 or β2 adrenergic receptors, ELF-PEMF exposure induced a rapid and selective internalization of β2 adrenergic receptor. The β-adrenergic signaling, was reduced trough Gi protein by ELF-PEMF exposure since the phosphorylation level of phospholamban and the PI3K pathway were impaired after isoproterenol stimulation in neonatal cardiomyocytes. Long term effects of ELF-PEMF exposure were assessed in cultures of isolated cardiomyocytes. ELF-PEMF counteracts cell size increase, the generation of binucleated of cardiomyocytes and prevents the up-regulation of hypertrophic markers after β-adrenergic stimulation, indicating an inhibition of cell growth and maturation. These data show that short and long term exposure to ELF-PEMF induces a reduction of cardiac β-adrenergic response at molecular, functional and adaptative levels.

Transient receptor potential vanilloid type-1 (TRPV1) channels expressed in primary afferent neurons play a critical role in nociception triggered by endogenous and exogenous compounds. In the present study, the functional and biochemical interaction between TRPV1 channels and type-α peroxisome proliferator-activated receptors (PPARα) has been investigated. In TRPV1-expressing CHO cells, patch-clamp studies revealed that acute application of the PPARα agonists clofibrate (CLO; 0.1-100 μM), WY14643 (1-300 μM), or GW7647 (0.1-100 nM) activated TRPV1 currents in a concentration-dependent manner, with EC50s of 5.3 ± 0.8 μM, 13.0 ± 1.2 μM, and 12.7 ± 0.3 nM, respectively. The role of PPARα in these pharmacological responses was confirmed by the ability of the PPARα antagonist GW6471 (10 μM) to block CLO-, WY14643- and GW7647-induced TRPV1 activation, and by the observation that modulation of PPARα levels via siRNA-mediated suppression or PPARα over-expression affected TRPV1 channel activation by PPARα agonists accordingly. In cells cotransfected with PPARα and TRPV1, PPARα receptors were detected in TRPV1-immunoprecipitated fractions. When compared to capsaicin (CAP), TRPV1 currents activated by PPARα agonists showed a higher degree of acute desensitization and tachyphylaxis; moreover, GW7647, when pre-incubated at a concentration (1nM) unable to activate TRPV1 currents per se, desensitized CAP-induced TRPV1 currents. Finally, a sub-effective concentration of each PPARα agonist inhibited TRPV1-dependent bradykinin-induced [Ca(2+)]i transients in sensory neurons. Collectively, these results provide evidence for a PPARα-mediated pathway triggering TRPV1 channel activation and desensitization, and highlight a novel mechanism which might contribute to the analgesic effects shown by PPARα agonists in vivo.

The exceptional versatility of calmodulin (CaM) three-dimensional arrangement is reflected in the growing number of structural models of CaM/protein complexes currently available in the Protein Data Bank (PDB) database, revealing a great diversity of conformations, domain organization, and structural responses to Ca(2+). Understanding CaM binding is complicated by the diversity of target proteins sequences. Data mining of the structures shows that one face of each of the eight CaM helices can contribute to binding, with little overall difference between the Ca(2+) loaded N- and C-lobes and a clear prevalence of the C-lobe low Ca(2+) conditions. The structures reveal a remarkable variety of configurations where CaM binds its targets in a preferred orientation that can be reversed and where CaM rotates upon Ca(2+) binding, suggesting a highly dynamic metastable relation between CaM and its targets. Recent advances in structure-function studies and the discovery of CaM mutations being responsible for human diseases, besides expanding the role of CaM in human pathophysiology, are opening new exciting avenues for the understanding of the how CaM decodes Ca(2+)-dependent and Ca(2+)-independent signals.

Modulation of the transient receptor potential melastatin type-8 (TRPM8), the receptor for menthol acting as the major sensor for peripheral innocuous cool temperatures, has several important applications in pharmaceutical, food and cosmetic industries. In the present study, we designed 12 isoxazole derivatives and tested their pharmacological properties both in F11 sensory neurons in vitro , and in an in vivo model of cold allodynia. In F11 sensory neurons, single-cell Ca 2+ -imaging experiments revealed that, when compared to menthol, some newly-synthesized compounds were up to 200-fold more potent, though none of them showed an increased efficacy. Some isoxazole derivatives potentiated allodynic responses elicited by acetone when administered to rats subjected to sciatic nerve ligation; when compared to menthol, these compounds were efficacious at earlier (0–2 min) but not later (7–9 or 14–16 min) time points. Docking experiments performed in a human TRPM8 receptor model revealed that newly-synthesized compounds might adopt two possible conformations, thereby allowing to distinguish “menthol-like” compounds (characterized by high efficacy/low potency), and “icillin-like” compounds (with high potency/low efficacy). Collectively, these data provide rationale structure–activity relationships for isoxazole derivatives acting as TRPM8 agonists, and suggest their potential usefulness for cold-evoked analgesia.

Background There is no doubt that future discoveries in the field of biochemistry will depend on the implementation of novel biosensing techniques, able to record biophysiological events with minimal biological interference. In this respect, organic electronics may represent an important new tool for the analysis of structures ranging from single molecules up to cellular events. Specifically, organic field-effect transistors (OFET) are potentially powerful devices for the real-time detection/transduction of bio-signals. Despite this interest, up to date, the experimental data useful to support the development of OFET-based biosensors are still few and, in particular, n-type (electron-transporting) devices, being fundamental to develop highly-performing circuits, have been scarcely investigated. Methods Here, films of N,N′-1H,1H-perfluorobutyldicyanoperylene-carboxydi-imide (PDIF-CN2) molecules, a recently-introduced and very promising n-type semiconductor, have been evaporated on glass and silicon dioxide substrates to test the biocompatibility of this compound and its capability to stay electrically-active even in liquid environments. Results We found that PDIF-CN2 transistors can work steadily in water for several hours. Biocompatibility tests, based on in-vitro cell cultivation, remark the need to functionalize the PDIF-CN2 hydrophobic surface by extra-coating layers (i.e. poly- l -lysine) to favor the growth of confluent cellular populations. Conclusions Our experimental data demonstrate that PDIF-CN2 compound is an interesting organic semiconductor to develop electronic devices to be used in the biological field. General significance This work contributes to define a possible strategy for the fabrication of low-cost and flexible biosensors, based on complex organic complementary metal-oxide-semiconductor (CMOS) circuitry including both p- (hole-transporting) and n-type transistors. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.

The K(v)7.2 subunits are the main molecular determinants of the M-current, a widespread K(+) current regulating neuronal excitability. Mutations in the K(v)7.2 gene cause benign familial neonatal seizures, an autosomally inherited human epilepsy. The benign familial neonatal seizure-causing mutations include those at arginine residues at positions 207 and 214 in the S(4) segment of K(v)7.2. In this study, each of the six S(4) arginines was individually replaced with neutral glutamines, and the functional properties of mutant channels were studied by whole-cell and single-channel voltage-clamp measurements. The results obtained suggest that each S(4) arginine residue plays a relevant role in the voltage-dependent gating of K(v)7.2 channels. In particular, a decreased positive charge at the N-terminal end of S(4) stabilized the activated state of the voltage-sensor, whereas positive-charge neutralization at the C-terminal end of S(4) favored the resting conformation. Strikingly, neutralization of a single arginine at position 201 was sufficient to cause a significant loss of voltage dependence in channel activation. Moreover, by comparing the functional properties of glutamine versus tryptophan substitution, we found steric bulk to play a relevant role at position 207, but not at position 214, in which the main functional effect of this disease-causing mutation seems to be a consequence of the loss of the positive charge.

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.

Corrigendum to “Neutralization of a unique, negatively-charged residue in the voltage sensor of KV7.2 subunits in a sporadic case of benign familial neonatal seizures” [Neurobiol. Dis. 34 (2009) 501–510] Francesco Miceli , Maria Virginia Soldovieri , Licia Lugli , Giulia Bellini , Paolo Ambrosino , Michele Migliore , Emanuele Miraglia del Giudice , Fabrizio Ferrari , Antonio Pascotto , Maurizio Taglialatela a,b,⁎