A dual voltage clamp technique to study gap junction hemichannels in astrocytes cultured from neonatal rodent spinal cords

Astrocytes express surface channels involved in purinergic signaling, and among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) mediate ATP release that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, i.e., open and close, under physiological and pathological conditions, single channel conductance of Px1 HCs is not well defined. Here, we developed a dual voltage clamp technique in HeLa cells overexpressing human Px1-YFP, and then applied this system to rodent spinal astrocytes. Single channels were recorded in cell attached patches and evoked with ramp cycles of 2 s duration and -/+ 80-100 mV amplitude or rectangular pulses through another pipette in whole cell clamp. Conductance of Px1 HC openings recorded during ramp stimuli ranged 25-110 pS. Based on their single channel conductances, Px1 HCs could be distinguished from Cx43 HCs and P2X7 receptors (P2X7Rs) in spinal astrocytes during dual voltage clamp experiments. Furthermore, we found that single channel activity of Cx43 HCs and P2X7Rs was increased, and that of Px1 HCs was decreased, in spinal astrocytes treated for 7h with FGF-1, a growth factor implicated in neurodevelopment, repair and inflammation.

High-throughput Evaluation of Epilepsy-associated KCNQ2 Variants Reveals Functional and Pharmacological Heterogeneity

Hundreds of KCNQ2 variants have been identified by genetic testing of children with early onset epilepsy and/or developmental disability. Voltage-clamp recording from heterologous cells has proved useful for establishing deleterious functional effects of KCNQ2 variants, but procedures adapting these assays for standardized, higher throughput data collection and reporting are lacking. In this study, we employed automated patch clamp recording to assess in parallel the functional and pharmacological properties of 79 missense and 2 in-frame deletion variants of KCNQ2. Among the variants we studied were a training set of 18 pathogenic variants previously studied by voltage-clamp recording, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. Variant KCNQ2 subunits were transiently expressed in a cell line stably expressing KCNQ3 to reconstitute the physiologically relevant channel complex. Variants with severe loss-of-function were also co-expressed 1:1 with WT KCNQ2 in the KCNQ3 cell line to mimic the heterozygous genotype and assess dominant-negative behavior. In total, we analyzed electrophysiological data recorded from 9,480 cells. The functional properties of WT KCNQ2/KCNQ3 channels and pharmacological responses to known blockers and activators determined by automated patch clamp recording were highly concordant with previous findings. Similarly, functional properties of 18 known pathogenic variants largely matched previously published results and the validated automated patch clamp assay. Many of the 39 previously unstudied disease-associated KCNQ2 variants exhibited prominent loss-of-function and dominant-negative effects, providing strong evidence in support of pathogenicity. All variants, exhibit response to retigabine (10 μM), although there were differences in maximal responses. Variants within the ion selectivity filter exhibited the weakest responses whereas retigabine had the strongest effect on gain-of-function variants in the voltage-sensor domain. Our study established a high throughput method to detect deleterious functional consequences of KCNQ2 variants. We demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants but this does not occur with rare population variation in this gene. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine.

Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current

The cardiac Mg2+-sensitive, TRPM6, and TRPM7-like channels remain undefined, especially with the uncertainty regarding TRPM6 expression in cardiomyocytes. Additionally, their contribution to the cardiac action potential (AP) profile is unclear. Immunofluorescence assays showed the expression of the TRPM6 and TRPM7 proteins in isolated pig atrial and ventricular cardiomyocytes, of which the expression was modulated by incubation in extracellular divalent cation-free conditions. In patch clamp studies of cells dialyzed with solutions containing zero intracellular Mg2+ concentration ([Mg2+]i) to activate the Mg2+-sensitive channels, raising extracellular [Mg2+] ([Mg2+]o) from the 0.9-mM baseline to 7.2 mM prolonged the AP duration (APD). In contrast, no such effect was observed in cells dialyzed with physiological [Mg2+]i. Under voltage clamp, in cells dialyzed with zero [Mg2+]i, depolarizing ramps induced an outward-rectifying current, which was suppressed by raising [Mg2+]o and was absent in cells dialyzed with physiological [Mg2+]i. In cells dialyzed with physiological [Mg2+]i, raising [Mg2+]o decreased the L-type Ca2+ current and the total delayed-rectifier current but had no effect on the APD. These results suggest a co-expression of the TRPM6 and TRPM7 proteins in cardiomyocytes, which are therefore the molecular candidates for the native cardiac Mg2+-sensitive channels, and also suggest that the cardiac Mg2+-sensitive current shortens the APD, with potential implications in arrhythmogenesis.

An Improved Voltage Clamp Circuit Suitable for Accurate Measurement of the Conduction Loss of Power Electronic Devices

Power electronic devices are essential components of high-capacity industrial converters. Accurate assessment of their power loss, including switching loss and conduction loss, is essential to improving electrothermal stability. To accurately calculate the conduction loss, a drain–source voltage clamp circuit is required to measure the on-state voltage. In this paper, the conventional drain–source voltage clamp circuit based on a transistor is comprehensively investigated by theoretical analysis, simulations, and experiments. It is demonstrated that the anti-parallel diodes and the gate-shunt capacitance of the conventional drain–source voltage clamp circuit have adverse impacts on the accuracy and security of the conduction loss measurement. Based on the above analysis, an improved drain–source voltage clamp circuit, derived from the conventional drain–source voltage clamp circuit, is proposed to solve the above problems. The operational advantages, physical structure, and design guidelines of the improved circuit are fully presented. In addition, to evaluate the influence of component parameters on circuit performance, this article comprehensively extracts three electrical quantities as judgment indicators. Based on the working mechanism of the improved circuit and the indicators mentioned above, general mathematical analysis and derivation are carried out to give guidelines for component selection. Finally, extensive experiments and detailed analyses are presented to validate the effectiveness of the proposed drain–source voltage clamp circuit. Compared with the conventional drain–source voltage clamp circuit, the improved drain–source voltage clamp circuit has higher measurement accuracy and working security when measuring conduction loss, and the proposed component selection method is verified to be reasonable and effective for better utilizing the clamp circuit.

Plant “helper” immune receptors are Ca2+-permeable nonselective cation channels

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. In Arabidopsis, a subfamily of “helper” NLRs are required by many “sensor” NLRs. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta and conferred cytoplasmic Ca2+ influx in plant and human cells. NRG1.1-dependent Ca2+ influx and cell death were sensitive to Ca2+ channel blockers and were suppressed by mutations impacting oligomerization or plasma membrane enrichment. Ca2+ influx and cell death mediated by NRG1.1 and ACTIVATED DISEASE RESISTANCE 1 (ADR1), another “helper” NLR, required conserved negatively charged N-terminal residues. Whole-cell voltage-clamp recordings demonstrate that Arabidopsis “helper” NLRs form Ca2+-permeable cation channels to directly regulate cytoplasmic Ca2+ levels and consequent cell death. Thus, “helper” NLRs transduce cell death signals directly.

Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization

Gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of rat P2X2 during ATP- and voltage-dependent gating, using a voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field. We also observed slow and voltage-dependent F changes at Ala337, which reflect structural rearrangements. Furthermore, we determined that the interaction between Ala337 in TM2 and Phe44 in TM1, which are in close proximity in the ATP-bound open state, is critical for activation. Taking these results together, we propose that the voltage dependence of the interaction within the converged electric field underlies the voltage-dependent gating.

Calcium/calmodulin-dependent protein kinase II stimulates the inward rectifier potassium current in beta-adrenergic adaptation of ventricular cardiomyocytes

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): NEW NATIONAL EXCELLENCE PROGRAM OF THE MINISTRY FOR INNOVATION AND TECHNOLOGY FROM THE SOURCE OF THE NATIONAL RESEARCH, DEVELOPMENT AND INNOVATION FUND. Introduction and purpose Acute β-adrenergic receptor (β-AR) stimulation shortens the ventricular action potential (AP). This effect is mainly regulated by the β- adrenergic stimulation of the cardiac potassium currents. Our aim was to investigate the extent of calcium/calmodulin-dependent protein kinase II (CaMKII) involvement in mediating the effect of β-AR activation on the inward rectifier potassium current – I K1 . Methods We carried out our experiments on isolated cardiomyocytes originating from canine left ventricles. The inward rectifier potassium current – I K1 was measured under a "canonical" AP under action potential voltage clamp conditions. Data were collected in four study groups [1] Control conditions (CTRL) [2] Inhibition of CaMKII with 1 µM KN-93 (KN-93) [3] Inhibition of PKA with 3 µM H-89 (H-89) [4] Acute β-adrenergic stimulation with 10 nM isoproterenol (ISO) [5] β-adrenergic stimulation with CaMKII inhibition (KN-93 + ISO) [6] β-adrenergic stimulation with PKA inhibition (H-89 + ISO) [7] β-adrenergic stimulation with inhibited PKA and CaMKII (KN-93 + H-89 + ISO) Results I K1 current amplitude did not differ among the studied groups, the total carried charge however was significantly, about 30 % larger in the ISO group compared to CTRL, and about 20 % larger compared to KN-93 + ISO. Under beta- adrenergic stimulation, I K1 starts to activate earlier during the AP plateau. I K1 density was about 3 times greater both at +20 mV and at 0 mV membrane potential under the command "canonical" AP in ISO compared to CTRL. Similarly, I K1 density was about 60 % and 90 % larger at +20 mV and at 0 mV, respectively, in KN-93 + ISO compared to KN-93. Similar results have been obtained by conventional voltage-clamp technique. Conclusion Based on the results of our researches the CaMKII activation plays an important role in β -adrenergic stimulation the I K1 potassium current.