Nitrergic modulation of neuronal excitability in the mouse hippocampus is mediated via regulation of Kv2 and voltage-gated sodium channels.

Regulation of neuronal activity is a necessity for communication and information transmission. Many regulatory processes which have been studied provide a complex picture of how neurons can respond to permanently changing functional requirements. One such activity-dependent mechanism involves signaling mediated by nitric oxide (NO). Within the brain, NO is generated in response to neuronal NO synthase (nNOS) activation but NO-dependent pathways regulating neuronal excitability in the hippocampus remain to be fully elucidated. This study was set out to systematically assess the effects of NO on ion channel activities and intrinsic excitabilities of pyramidal neurons within the CA1 region of the mouse hippocampus. We characterized whole-cell potassium and sodium currents, both involved in action potential (AP) shaping and propagation and determined NO-mediated changes in excitabilities and AP waveforms. Our data describe a novel signaling by which NO, in a cGMP-independent manner, suppresses voltage-gated Kv2 potassium and voltage-gated sodium channel activities, thereby widening AP waveforms and reducing depolarization-induced AP firing rates. Our data show that glutathione, which possesses denitrosylating activity, is sufficient to prevent the observed nitrergic effects on potassium and sodium channels, whereas inhibition of cGMP signaling is also sufficient to abolish NO modulation of sodium currents. We propose that NO suppresses both ion channel activities via redox signaling and that an additional cGMP-mediated component is required to exert effects on sodium currents. Both mechanisms result in a dampened excitability and firing ability providing new data on nitrergic activities in the context of activity-dependent regulation of neuronal function following nNOS activation.

Biophysical, Molecular, and Pharmacological Characterization of Voltage-Dependent Sodium Channels From Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND: The ability to differentiate patient-specific human induced pluripotent stem cells in cardiac myocytes (hiPSC-CM) offers novel perspectives for cardiovascular research. A number of studies, that reported mainly on current-voltage curves used hiPSC-CM to model voltage-gated Na+ channel (Nav) dysfunction. However, the expression patterns and precise biophysical and pharmacological properties of Nav channels from hiPSC-CM remain unknown. Our objective was to study the characteristics of Nav channels from hiPSC-CM and assess the appropriateness of this novel cell model. METHODS: We generated hiPSC-CM using the recently described monolayer-based differentiation protocol. RESULTS: hiPSC-CM expressed cardiac-specific markers, exhibited spontaneous electrical and contractile activities, and expressed distinct Nav channels subtypes. Electrophysiological, pharmacological, and molecular characterizations revealed that, in addition to the main Nav1.5 channel, the neuronal tetrodotoxin (TTX)-sensitive Nav1.7 channel was also significantly expressed in hiPSC-CM. Most of the Na+ currents were resistant to TTX block. Therapeutic concentrations of lidocaine, a class I antiarrhythmic drug, also inhibited Na+ currents in a use-dependent manner. Nav1.5 and Nav1.7 expression and maturation patterns of hiPSC-CM and native human cardiac tissues appeared to be similar. The 4 Navß regulatory subunits were expressed in hiPSC-CM, with ß3 being the preponderant subtype. CONCLUSIONS: The findings indicated that hiPSC-CM robustly express Nav1.5 channels, which exhibited molecular and pharmacological properties similar to those in native cardiac tissues. Interestingly, neuronal Nav1.7 channels were also expressed in hiPSC-CM and are likely to be responsible for the TTX-sensitive Nav current.

Evaluation of microtransplantation of rat brain neurolemma into Xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage-sensitive ion channels.

Microtransplantation of mammalian brain neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Nav1.1-Nav1.9) was determined in neurolemma using automated western blotting, with the predominant isoforms expressed being Nav1.2 and Nav1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE was without effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs in Xenopus oocytes and with many other electrophysiological approaches, validating the use of the microtransplantation procedure as a toxicologically-relevant ex vivo assay. Once fully characterized, it is likely that this approach could be expanded to study the role of environmental toxicants and contaminants on various target tissues (e.g. neural, reproductive, developmental) from many species.