Whole-cell Configuration Research Articles

The Roles of BK Potassium Channels in Neuronal Excitability Differ in Excitatory and Inhibitory Hippocampal Neurons During Early Development

BK potassium channels are are activated by intracellular Ca+ and depolarization, opening during action potential firing and speeding repolarization. Because of this, they are key regulators of fast spiking activity and important determinants of neuronal excitability. Due to their role in regulating excitability as well as their broad expression in both excitable and non‐excitable tissues, BK channels are implicated in many pathological states including epilepsy. The BK channel antagonist, paxilline, has been shown to delay seizure induction in adult rodents that have had prior seizures, but not in seizure naïve rodents. History of seizures has been shown induce expression of more Ca2+ sensitive BK channel splice variants, which may underlie the difference in paxilline efficacy between the two states. Because expression of more Ca2+ sensitive isoforms predominates in neonatal rodents ‐similar to in the epileptic adults, we hypothesize that paxilline may be an effective inhibitor of seizure activity even in seizure‐naïve neonatal rodents. Our first goal is to understand how developmental shifts in BK channel expression and function map to changes in neuronal and circuit excitability. Our second goal is to test whether BK channel blockade can modulate seizure activity in neonates, as there is a need to identify pharmacological targets for seizures in neonates and children, which are often intractable to conventional treatments.To test how BK channel function changes with age and by cell type we measured the effect of paxilline on action potential (AP) firing ‐both on single spike kinetics and prolonged firing‐ in two classes of hippocampal neurons, CA1 pyramidal neurons and parvalbumin interneurons (PVIns), a key inhibitory component of local hippocampal circuits. We used P4‐5, P9‐10, and P14‐15 C57BL/6J mice with labelled PVIns. We also tested the efficacy of paxilline in reducing hyperexcitability evoked by application of 0 Mg2+ aCSF in brain slices, an established seizure model.We patched pyramidal neurons and PVIns in whole‐cell current‐clamp configuration and evoked APs using square depolarizing pulses. BK channel blockade with 1mM paxilline lengthens duration of APs in P4‐5 and P9‐10 pyramidal neurons and P4‐5 PVIns but does not affect P9‐10 PVIn AP duration (paired t‐tests, p<0.05). While BK blockade has the least effect on single APs in the P9‐10 PVIns, it has the most effect on the input‐output relationship and spike‐frequency accommodation in these neurons (3‐way ANOVA, p<0.001 for interaction of age and cell type in input‐output; 3‐way ANOVA p<0.05 for main effect of age in frequency adaptation).We measured the effect of BK channel blockade in controlling hyperexcitability in P4‐15 mice. We found that 0 Mg2+ aCSF application induced spontaneous AP firing in patched hippocampal pyramidal neurons within 3.86±0.87 minutes but when 0 Mg2+ aCSF was coapplied with 1 mM paxilline the average onset of spontaneous activity occurred at 8.43±1.40 minutes (t‐test p<0.05).For the first goal, we show that BK channels’ relative importance in regulating AP firing changes with age and by neuronal population. Toward the second goal we report that BK channel blockade can reduce hyperexcitability in a brain slice model of epilepsy.

Bi‐directional pH dependency of CALHM1 ion channel

Calcium homeostasis modulator (CALHM) membrane proteins are nonselective ion channels drawing attention regarding their roles in modulating neuronal activity and taste sensation. CALHM1‐expressing cells show voltage‐gated slowly activating ionic currents. The voltage‐dependence of CALHM1 is negatively affected by extracellular Ca2+ ([Ca2+]e), while facilitated by temperature. Here we investigated the effect of extracellular and intracellular pH (pHe and pHi) on the electrophysiological properties of CALHM1 overexpressed in CHO cells. When normalized to the control at pHe 7.4, the amplitudes of CALHM1 current (ICALHM1) were suppressed to 20 % at pHe 6.2, while increased to 500 % at pHe 8.6. In the whole‐cell configuration of patch clamp, changing pHi from 7.4 to 6.2 or 8.6 showed the acid‐induced inhibition and alkali‐induced activation of ICALHM1. The voltage‐dependence of CALHM1 was shifted to the negative membrane potential at the alkaline pHe and pHi, while positive membrane potential at the acidic pHe and pHi. The pHi effect was also confirmed in the inside‐out configuration of patch clamp. The open probability (Po) of multiple CALHM1 channel decreased at pH 6.2, while increased at pH 8.6 compared with pH 7.4. To get a clue to identify the pH sensing amino acid residues, we utilized homology model performed with the SWISS‐MODEL workspace, based on the cryo‐EM structure of human CALHM2. Then we conducted site‐directed mutagenesis of the water‐accessible charged amino acids of CALHM1. Among 23 candidates, mutations at E17, K229, E233, D257 and E259 in the intracellular space reduced the alkali‐induced facilitation of ICALHM1, while not altered the acid‐induced inhibition. According to the molecular structure model of killifish CALHM1 with octameric structure, the five residues located at N or C termini gathered closely in the intracellular space. Considering the large pore diameter of CALHM1 (~14 Å), we cautiously propose that the sensitivity to alkaline pHe could be because the protons transfer between hydronium ions in the pore. The study firstly demonstrated the remarkable pH‐sensitivity of CALHM1 and its pH‐sensing residues, which might be responsible for the modulation of neuronal excitability during dynamic changes of the regional pH in brain or sensory organs.

Deficiency of Thyroid Hormone Reduces Voltage-Gated Na+ Currents as Well as Expression of Na+/K+-ATPase in the Mouse Hippocampus.

Mice lacking functional thyroid follicular cells, Pax8−/− mice, die early postnatally, making them suitable models for extreme hypothyroidism. We have previously obtained evidence in postnatal rat neurons, that a down-regulation of Na+-current density could explain the reduced excitability of the nervous system in hypothyroidism. If such a mechanism underlies the development of coma and death in severe hypothyroidism, Pax8−/− mice should show deficits in the expression of Na+ currents and potentially also in the expression of Na+/K+-ATPases, which are necessary to maintain low intracellular Na+ levels. We thus compared Na+ current densities in postnatal mice using the patch-clamp technique in the whole-cell configuration as well as the expression of three alpha and two beta-subunits of the Na+/K+-ATPase in wild type versus Pax8−/− mice. Whereas the Na+ current density in hippocampal neurons from wild type mice was upregulated within the first postnatal week, the Na+ current density remained at a very low level in hippocampal neurons from Pax8−/− mice. Pax8−/− mice also showed significantly decreased protein expression levels of the catalytic α1 and α3 subunits of the Na+/K+-ATPase as well as decreased levels of the β2 isoform, with no changes in the α2 and β1 subunits.

The role of activation of two different sGC binding sites by NO-dependent and NO-independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes.

The mechanoelectrical feedback (MEF) mechanism in the heart that plays a significant role in the occurrence of arrhythmias, involves cation flux through cation nonselective stretch‐activated channels (SACs). It is well known that nitric oxide (NO) can act as a regulator of MEF. Here we addressed the possibility of SAC’s regulation along NO‐dependent and NO‐independent pathways, as well as the possibility of S‐nitrosylation of SACs. In freshly isolated rat ventricular cardiomyocytes, using the patch‐clamp method in whole‐cell configuration, inward nonselective stretch‐activated cation current ISAC was recorded through SACs, which occurs during dosed cell stretching. NO donor SNAP, α1‐subunit of sGC activator BAY41‐2272, sGC blocker ODQ, PKG blocker KT5823, PKG activator 8Br‐cGMP, and S‐nitrosylation blocker ascorbic acid, were employed. We concluded that the physiological concentration of NO in the cell is a necessary condition for the functioning of SACs. An increase in NO due to SNAP in an unstretched cell causes the appearance of a Gd3+‐sensitive nonselective cation current, an analog of ISAC , while in a stretched cell it eliminates ISAC . The NO‐independent pathway of sGC activation of α subunit, triggered by BAY41‐2272, is also important for the regulation of SACs. Since S‐nitrosylation inhibitor completely abolishes ISAC , this mechanism occurs. The application of BAY41‐2272 cannot induce ISAC in a nonstretched cell; however, the addition of SNAP on its background activates SACs, rather due to S‐nitrosylation.ODQ eliminates ISAC , but SNAP added on the background of stretch increases ISAC in addition to ODQ. This may be a result of the lack of NO as a result of inhibition of NOS by metabolically modified ODQ. KT5823 reduces PKG activity and reduces SACs phosphorylation, leading to an increase in ISAC . 8Br‐cGMP reduces ISAC by activating PKG and its phosphorylation. These results demonstrate a significant contribution of S‐nitrosylation to the regulation of SACs.

L-cysteine modulates visceral nociception mediated by the CaV2.3 R-type calcium channels

CaV2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant CaV2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant CaV2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of CaV2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of CaV2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.

Cardioprotective effects of N-methylacetazolamide mediated by inhibition of L-type Ca2+ channel current

Our objective was to examine the effects of N-methylacetazolamide (NMA), a non‑carbonic anhydrase inhibitor, on ischemia-reperfusion injury. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC):110 min of perfusion and 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R). Both groups were repeated in presence of NMA (5 μM), administered during the first 10 min of R. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function, respectively. The content of P-Akt, P-PKCε, P-Drp1 and calcineurin Aβ were measured. In cardiomyocytes the L-type Ca2+ current (ICaL) was recorded with the whole-cell configuration of patch-clamp technique. The addition of NMA to non-ischemic hearts decreased 15% the contractility. In ischemic hearts (IC group), NMA decreased IS (22 ± 2% vs 32 ± 2%, p < 0.05) and improved the post-ischemic recovery of myocardial function. At the end of R, LVDP was 54 ± 7% vs 18 ± 3% and LVEDP was 23 ± 8 vs. 55 ± 7 mmHg ¨p < 0.05¨. The level of P-Akt, P-PKCε and P-Drp1 increased and the expression of calcineurin Aβ decreased in NMA treated hearts. Peak ICaL density recorded at 0 mV was smaller in myocytes treated with NMA than in non-treated cells (−1.91 ± 0.15 pA/pF vs −2.32 ± 0.17 pA/pF, p < 0.05). These data suggest that NMA protects the myocardium against ischemia-reperfusion injury through an attenuation of mitochondrial fission by calcineurin/Akt/PKCε-dependent pathways associated to the decrease of ICaL current.

Effect of Biphasic kHz Field Stimulation on CA1 Pyramidal Neurons in Slices

Electrical stimulation in the kilohertz-frequency range has been successfully used for treatment of various neurological disorders. Nevertheless, the mechanisms underlying this stimulation are poorly understood. To study the effect of kilohertz-frequency electric fields on neuronal membrane biophysics we developed a reliable experimental method to measure responses of single neurons to kilohertz field stimulation in brain slice preparations. In the submerged brain slice pyramidal neurons of the CA1 subfield were recorded in the whole-cell configuration before, during and after stimulation with a biphasic charge balanced electric field at 2kHz, 5kHz or 10 kHz. The slice was placed in a 1 mm gap between two parallel 2 mm long platinum-iridium 0.1 mm diameter wire electrodes. Whole-cell recordings lasted for an hour or longer. Typically, a few 5-10 min long sessions of kHz-field stimulation (kHz-FS) with various frequency/amplitude combinations were applied to assess the type of neuronal response and possible changes of its membrane characteristics. It was found that kHz-field stimulation at all frequencies elicited reproducible excitatory neuronal responses lasting throughout stimulation period, but not after cessation of kHz-FS. During kHz-FS the rheobase usually decreased. In addition, spontaneous firing might be initiated in some silent neurons or became more intense in previously spontaneously active neurons. Response thresholds were in the range of 0.5-2 mA and were higher at higher frequencies. Blockade of glutamatergic synaptic transmission did not alter the magnitude of responses. Inhibitory synaptic input was not changed by kilohertz field stimulation. We conclude that kHz-frequency current applied in brain tissue has an excitatory effect on pyramidal neurons during stimulation. This effect is more prominent and occurs at a lower stimulus intensity at a frequency of 2kHz as compared to 5kHz and 10kHz.

Single-Walled Carbon Nanotubes Inhibit TRPC4-Mediated Muscarinic Cation Current in Mouse Ileal Myocytes.

Single-walled carbon nanotubes (SWCNTs) are characterized by a combination of rather unique physical and chemical properties, which makes them interesting biocompatible nanostructured materials for various applications, including in the biomedical field. SWCNTs are not inert carriers of drug molecules, as they may interact with various biological macromolecules, including ion channels. To investigate the mechanisms of the inhibitory effects of SWCNTs on the muscarinic receptor cation current (mICAT), induced by intracellular GTPγs (200 μM), in isolated mouse ileal myocytes, we have used the patch-clamp method in the whole-cell configuration. Here, we use molecular docking/molecular dynamics simulations and direct patch-clamp recordings of whole-cell currents to show that SWCNTs, purified and functionalized by carboxylation in water suspension containing single SWCNTs with a diameter of 0.5–1.5 nm, can inhibit mICAT, which is mainly carried by TRPC4 cation channels in ileal smooth muscle cells, and is the main regulator of cholinergic excitation–contraction coupling in the small intestinal tract. This inhibition was voltage-independent and associated with a shortening of the mean open time of the channel. These results suggest that SWCNTs cause a direct blockage of the TRPC4 channel and may represent a novel class of TRPC4 modulators.

Functional Analysis of SCN5A Genetic Variants Associated with Brugada Syndrome

Background: Brugada syndrome (BrS) is a rare inherited cardiac arrhythmia with increased risk of sudden cardiac death. Mutations in gene SCN5A, which encodes the α-subunit of cardiac voltage-gated sodium channel Na_V1.5, have been identified in over 20% of patients with BrS. However, only a small fraction of Na_V1.5 variants, which are associated with BrS, are characterized in electrophysiological experiments. Results: Here we explored variants V281A and L1582P, which were found in our patients with BrS, and variants F543L and K1419E, which are reportedly associated with BrS. Heterologous expression of the variants in CHO-K1 cells and the Western blot analysis demonstrated that each variant appeared at the cell surface. We further measured sodium current in the whole-cell voltage clamp configuration. Variant F543L produced robust sodium current with a hyperpolarizing shift in the voltage dependence of steady-state fast inactivation. Other variants did not produce detectable sodium currents, indicating a complete loss of function. In a recent cryoEM structure of the hNa_V1.5 channel, residues V281, K1419, and L1582 are in close contacts with residues whose mutations are reportedly associated with BrS, indicating functional importance of respective contacts. Conclusions: Our results support the notion that loss of function of Na_V1.5 or decrease of the channel activity is involved in the pathogenesis of BrS.

Electrophysiological Effects of the Transient Receptor Potential Melastatin 4 Channel Inhibitor (4-Chloro-2-(2-chlorophenoxy)acetamido) Benzoic Acid (CBA) in Canine Left Ventricular Cardiomyocytes.

Transient receptor potential melastatin 4 (TRPM4) plays an important role in many tissues, including pacemaker and conductive tissues of the heart, but much less is known about its electrophysiological role in ventricular myocytes. Our earlier results showed the lack of selectivity of 9-phenanthrol, so CBA ((4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) was chosen as a new, potentially selective inhibitor. Goal: Our aim was to elucidate the effect and selectivity of CBA in canine left ventricular cardiomyocytes and to study the expression of TRPM4 in the canine heart. Experiments were carried out in enzymatically isolated canine left ventricular cardiomyocytes. Ionic currents were recorded with an action potential (AP) voltage-clamp technique in whole-cell configuration at 37 °C. An amount of 10 mM BAPTA was used in the pipette solution to exclude the potential activation of TRPM4 channels. AP was recorded with conventional sharp microelectrodes. CBA was used in 10 µM concentrations. Expression of TRPM4 protein in the heart was studied by Western blot. TRPM4 protein was expressed in the wall of all four chambers of the canine heart as well as in samples prepared from isolated left ventricular cells. CBA induced an approximately 9% reduction in AP duration measured at 75% and 90% of repolarization and decreased the short-term variability of APD90. Moreover, AP amplitude was increased and the maximal rates of phase 0 and 1 were reduced by the drug. In AP clamp measurements, CBA-sensitive current contained a short, early outward and mainly a long, inward current. Transient outward potassium current (Ito) and late sodium current (INa,L) were reduced by approximately 20% and 47%, respectively, in the presence of CBA, while L-type calcium and inward rectifier potassium currents were not affected. These effects of CBA were largely reversible upon washout. Based on our results, the CBA induced reduction of phase-1 slope and the slight increase of AP amplitude could have been due to the inhibition of Ito. The tendency for AP shortening can be explained by the inhibition of inward currents seen in AP-clamp recordings during the plateau phase. This inward current reduced by CBA is possibly INa,L, therefore, CBA is not entirely selective for TRPM4 channels. As a consequence, similarly to 9-phenanthrol, it cannot be used to test the contribution of TRPM4 channels to cardiac electrophysiology in ventricular cells, or at least caution must be applied.

Muscimol Directly Activates the TREK-2 Channel Expressed in GABAergic Neurons through Its N-Terminus.

The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.

Defective Function of KCa3.1 Channels in Lysosomal Storage Disorders

Background: The calcium-calmodulin-gated KCa3.1 is considered a key regulator of membrane hyperpolarization, maintenance of cellular electrolyte balance, Ca2+-signalling, secretion of pro-inflammatory cytokines, neurodegeneration, pathological tissue remodelling. Osteonecrosis/-penia, vasculopathies, and neurodegeneration are chronic and progressive inflammatory complications of several inherited lysosomal storage diseases, such as Gaucher Disease (GD1/GD3), Fabry Disease(FD), and Niemann-Pick disease type C1 (NPC1). Here we hypothesized that KCa3.1 functions are impaired in a LSD-related fashion in patients' fibroblasts or the monocyte/macrophages lineage.Methods: A total of25 subjects were analyzed (5 FD, 5 NPC, 5 GD1, 5 GD2, 5 healthy controls). Fibroblasts from patients' skin biopsies were cultured in DMEM supplemented with 10% FCS (Biochrom KG, Berlin, Germany) and cells were used at passages 1-4. For patch-clamp experiments, cells were seeded on coverslips and used within 24 h. Monocytes isolated and purified from peripheral blood from age-matched GD patients (GD1 and GD3) and healthy volunteers (Ctrl). Measurements were repeated in patient's and Ctrl's macrophages that were differentiated from monocyte precursors in vitro by exposure to patient's or Ctrl's erythrocyte lysates for 4 days, respectively.Ca2+-activated K+ currents were measured in the whole-cell configuration using an EPC10-USB amplifier (HEKA, Electronics, Lambrecht-Pfalz, Germany) and a K pipette solution (intracellular) containing 1 μM Ca2+ free (in mM): 140 KCl, 1 MgCl2, 2 EGTA, 1.71 CaCl2 (1 μM [Ca2+]free), and 5 HEPES (adjusted to pH 7.2 with KOH). For data acquisition and analysis we used the patch-master program (HEKA). Ohmic leak currents of up to 1 nS were subtracted where appropriate. We quantified outward currents at a 0 mV holding potential (to avoid contamination with non-selective cation currents and chloride currents). Capacitance values (a measure of cell surface and thus cell size)Results: Patch-clamp demonstrated lower fibroblast KCa3.1 membrane functions in NPC1 and male FD patients. KCa3.1-currents in monocytes were similar in GD-1/3 and Ctrls. In differentiated and activated macrophages, the overall larger KCa3.1 currents in GD-1 macrophages were reduced to 40% of controls while in GD-3 macrophages KCa3.1 currents were barely detectable. Glucosylceramide was found to produce KCa3.1-current desensitization. At the gene level, KCa3.1-mRNA expression was impaired in NPC1, GD1/3, and male FD patients.Conclusions: Defective KCa3.1 functions are a feature and biomarker of cellular dysfunction in the LSD, GD1/3, FD, and NPC1 and supports the concept that biased lipid metabolism harms ion channel functions. Since KCa3.1 is involved in the mechanism of cellular differentiation, neurodegeneration, and inflammation, the impaired KCa3.1 -activity could be of pathomechanistic relevance in LSD cellular malfunction by promoting differentiation into pro-inflammatory cell types. Pharmacological targeting of KCa3.1 could be therapeutic utility to achieve neuroprotection in GD3 and NPC1 patients and bone complications in GD1. DisclosuresNo relevant conflicts of interest to declare.

Analysis of neuronal Ca2+ handling properties by combining perforated patch clamp recordings and the added buffer approach

Ca2+ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca2+. Intracellular Ca2+ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca2+ handling properties by combining the ‘added buffer’ approach [1] with perforated patch-clamp recordings [2]. Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca2+ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca2+ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca2+ handling properties, including immobile as well as mobile Ca2+ buffers.

SARS‐CoV‐2: pushing the E(nvelope)

SARS‐CoV‐2: pushing the E(nvelope)

Transfection methods for high-throughput cellular assays of voltage-gated calcium and sodium channels involved in pain.

Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, CaV2.2 and NaV1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPRTetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPRTetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both CaV2.2 and NaV1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells.

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

Automatic deep learning-driven label-free image-guided patch clamp system

Patch clamp recording of neurons is a labor-intensive and time-consuming procedure. Here, we demonstrate a tool that fully automatically performs electrophysiological recordings in label-free tissue slices. The automation covers the detection of cells in label-free images, calibration of the micropipette movement, approach to the cell with the pipette, formation of the whole-cell configuration, and recording. The cell detection is based on deep learning. The model is trained on a new image database of neurons in unlabeled brain tissue slices. The pipette tip detection and approaching phase use image analysis techniques for precise movements. High-quality measurements are performed on hundreds of human and rodent neurons. We also demonstrate that further molecular and anatomical analysis can be performed on the recorded cells. The software has a diary module that automatically logs patch clamp events. Our tool can multiply the number of daily measurements to help brain research.

Two-Photon Photoactivation of Rubi-Gaba for Studying the Role of the Antisecretory Factor in the Modulation of the GABAA Receptor in Rat Cerebellar Granule Cells In Vitro

Caged compounds, widely diffused in neurophysiology, are molecules biologically or functionally inert until a short pulse of light enables them. Illumination releases the caged effector allowing the activation, manipulation, and control of selective biological functions and variations. in particular, 3D two-photon excitation microscopy, coupled with the electrophysiological technique of the patch-clamp in whole-cell configuration, provides a useful tool for studying several neurobiological processes such as the localized, accurate, and specific receptor response of a well-defined neuronal area.

KV1.5-KVβ1.3 Recycling Is PKC-Dependent.

KV1.5 channel function is modified by different regulatory subunits. KVβ1.3 subunits assemble with KV1.5 channels and induce a fast and incomplete inactivation. Inhibition of PKC abolishes the KVβ1.3-induced fast inactivation, decreases the amplitude of the current KV1.5–KVβ1.3 and modifies their pharmacology likely due to changes in the traffic of KV1.5–KVβ1.3 channels in a PKC-dependent manner. In order to analyze this hypothesis, HEK293 cells were transfected with KV1.5–KVβ1.3 channels, and currents were recorded by whole-cell configuration of the patch-clamp technique. The presence of KV1.5 in the membrane was analyzed by biotinylation techniques, live cell imaging and confocal microscopy approaches. PKC inhibition resulted in a decrease of 33 ± 7% of channels in the cell surface due to reduced recycling to the plasma membrane, as was confirmed by confocal microscopy. Live cell imaging indicated that PKC inhibition almost abolished the recycling of the KV1.5–KVβ1.3 channels, generating an accumulation of channels into the cytoplasm. All these results suggest that the trafficking regulation of KV1.5–KVβ1.3 channels is dependent on phosphorylation by PKC and, therefore, they could represent a clinically relevant issue, mainly in those diseases that exhibit modifications in PKC activity.

Advancing mechanobiology by performing whole-cell patch clamp recording on mechanosensitive cells subjected simultaneously to dynamic stretch events

SummaryA comprehensive understanding of mechano-electrical coupling requires continuous intracellular electrical recordings being performed on cells undergoing simultaneously in vivo like strain events. Here, we introduce a linear strain single-cell electrophysiology (LSSE) system that meets these requirements by delivering highly reproducible unidirectional strain events with magnitudes up to 12% and strain rates exceeding 200%s−1 to adherent cells kept simultaneously in whole-cell patch-clamp recording configuration. Proof-of-concept measurements with NIH3T3 cells demonstrate that stable recording conditions are maintained over tens of strain cycles at maximal amplitudes and strain rates thereby permitting a full electrophysiological characterization of mechanically activated ion currents. Because mechano-electrical responses to predefined strain patterns can be investigated using any adherent wild-type or genetically modified cell type of interest, the LSSE system offers the perspective of providing advanced insights into mechanosensitive ion channel function that can finally be compared quantitatively among different types of channels and cells.