Patch Pipette Research Articles

Abstract 12302: Na V 1.6 Dysregulation by Mutant Calmodulin Enhances Late Sodium Current in Human iPSC Cardiomyocytes and Promotes Arrhythmias in D96V Calmodulin-Expressing Transgenic Mice

Calmodulin (CaM) mutants are associated with severe arrhythmias (calmodulinopathy). While dysregulation of Ca 2+ handling proteins (RyR2, Ca V 1.2) are known mediators of calmodulinopathy, the role of Na + channels (Na V s) remains unclear. Interestingly, effects on Ca V 1.2 do not fully explain action potential (AP) prolongation by the long QT syndrome CaM mutant D96V. We investigated Na V dysregulation by D96V-CaM and its arrhythmogenic impact. In wild-type (WT) murine cardiomyocytes (CM) dialysis of D96V-CaM (6.5 μM) via a patch pipette prolonged AP, induced early and delayed afterdepolarizations (EADs and DADs) and increased Ca 2+ waves compared to WT-CaM. The effects of D96V-CaM were abrogated by a Na V 1.6 blocker, 4,9-anhydrotetrodotoxin (4,9ahTTX, 300 nM; Fig. 1A ). Dialysis of D96V-CaM dysregulated inactivation of 4,9ahTTX-sensitive Na + current (I Na ) promoting late I Na in WT murine CM ( Fig. 1B ) and in Chinese Hamster Ovary (CHO) cells expressing Na V 1.6 but not Na V 1.5. D96V-CaM, but not WT-CaM, also promoted late I Na in human induced pluripotent stem cell (iPSC) - derived ventricular myocytes, underscoring translational relevance ( Fig. 1C ). To examine the arrhythmia potential of Na V 1.6 dysregulation by D96V-CaM, we generated mice with cardiac-specific expression of D96V-CaM (cD96V; tgD96V-CaM x β-MHC-Cre). cD96V mice exhibited increased late I Na and Ca 2+ waves resembling those in D96V-CaM-dialyzed WT CMs. In vivo cD96V mice evidenced prolonged QTc intervals and ventricular tachycardia (VT) during bradycardia (carbachol 0.5 mg/kg intraperitoneal). Cardiac-specific Na V 1.6 knockout protected cD96V mice from impaired I Na inactivation and arrhythmogenic Ca 2+ waves on cellular level as well as long QT and VT in vivo ( Fig. 1D, E ). In summary, D96V-CaM promotes arrhythmias by dysregulating Na V 1.6 and thereby, facilitating aberrant Ca 2+ release. Importantly, Na V 1.6 may be a therapeutic target for management of CaM-related arrhythmias.

Glial decline and loss of homeostatic support rather than inflammation defines cognitive aging.

Glial decline and loss of homeostatic support rather than inflammation defines cognitive aging.

Piezo1 and BKCa channels in human atrial fibroblasts: interplay and remodelling in atrial fibrillation

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Research and Arts Baden-Württemberg (MWK-BW Sonderlinie Medizin) Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence. One of the important indicators for AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to such changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in their expression and activity associated with AF. Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or with sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC-signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel and its pharmacology indicated presence of ‘big potassium channels’, BKCa. In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of stretch-induced BKCa current. No co-immunoprecipitation of Piezo1 and BKCa was detected. Human atrial fibroblasts express functional Piezo1 and BKCa channels. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data show the presence and activity of Piezo1 and BKCa in human atrial fibroblasts and suggest an interplay between the two in the absence of direct physical interactions.

Piezo1 and BKCa channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation

AimsAtrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF. Methods and resultsUsing primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent ‘big potassium channels’ (BKCa). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. ConclusionsHuman atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins.

Traumatic Brain Injury Impairs Systemic Vascular Function Through Disruption of Inward-Rectifier Potassium Channels.

Trauma can lead to widespread vascular dysfunction, but the underlying mechanisms remain largely unknown. Inward-rectifier potassium channels (Kir2.1) play a critical role in the dynamic regulation of regional perfusion and blood flow. Kir2.1 channel activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid that is degraded by phospholipase A2 (PLA2) in conditions of oxidative stress or inflammation. We hypothesized that PLA2-induced depletion of PIP2 after trauma impairs Kir2.1 channel function. A fluid percussion injury model of traumatic brain injury (TBI) in rats was used to study mesenteric resistance arteries 24 h after injury. The functional responses of intact arteries were assessed using pressure myography. We analyzed circulating PLA2, hydrogen peroxide (H2O2), and metabolites to identify alterations in signaling pathways associated with PIP2 in TBI. Electrophysiology analysis of freshly-isolated endothelial and smooth muscle cells revealed a significant reduction of Ba2+-sensitive Kir2.1 currents after TBI. Additionally, dilations to elevated extracellular potassium and BaCl2- or ML 133-induced constrictions in pressurized arteries were significantly decreased following TBI, consistent with an impairment of Kir2.1 channel function. The addition of a PIP2 analog to the patch pipette successfully rescued endothelial Kir2.1 currents after TBI. Both H2O2 and PLA2 activity were increased after injury. Metabolomics analysis demonstrated altered lipid metabolism signaling pathways, including increased arachidonic acid, and fatty acid mobilization after TBI. Our findings support a model in which increased H2O2-induced PLA2 activity after trauma hydrolyzes endothelial PIP2, resulting in impaired Kir2.1 channel function.

Intermolecular Cooperative Gating in Piezo1 Channels

Mechanosensitive Piezo1 channels enable animal cells to sense and respond to changes in membrane tension. The tension-sensitivity of Piezo1 is often estimated using the gigaseal pressurization technique, wherein pulses of air pressure applied to the backside of a patch pipette in cell-attached mode to increase tension according to the Young-Laplace law while channel-mediated currents are simultaneously recorded. Generally, the pressure-sensitivity of Piezo1 is virtually always inferred from patches containing more than one channel.

Non-linear Conductance, Rectification, and Mechanosensitive Channel Formation of Lipid Membranes.

There is mounting evidence that lipid bilayers display conductive properties. However, when interpreting the electrical response of biological membranes to voltage changes, they are commonly considered as inert insulators. Lipid bilayers under voltage-clamp conditions display current traces with discrete conduction-steps, which are indistinguishable from those attributed to the presence of protein channels. In current-voltage (I-V) plots they may also display outward rectification, i.e., voltage-gating. Surprisingly, this has even been observed in chemically symmetric lipid bilayers. Here, we investigate this phenomenon using a theoretical framework that models the electrostrictive effect of voltage on lipid membranes in the presence of a spontaneous polarization, which can be recognized by a voltage offset in electrical measurements. It can arise from an asymmetry of the membrane, for example from a non-zero spontaneous curvature of the membrane. This curvature can be caused by voltage via the flexoelectric effect, or by hydrostatic pressure differences across the membrane. Here, we describe I-V relations for lipid membranes formed at the tip of patch pipettes situated close to an aqueous surface. We measured at different depths relative to air/water surface, resulting in different pressure gradients across the membrane. Both linear and non-linear I-V profiles were observed. Non-linear conduction consistently takes the form of outward rectified currents. We explain the conductance properties by two mechanisms: One leak current with constant conductance without pores, and a second process that is due to voltage-gated pore opening correlating with the appearance of channel-like conduction steps. In some instances, these non-linear I-V relations display a voltage regime in which dI/dV is negative. This has also been previously observed in the presence of sodium channels. Experiments at different depths reveal channel formation that depends on pressure gradients. Therefore, we find that the channels in the lipid membrane are both voltage-gated and mechanosensitive. We also report measurements on black lipid membranes that also display rectification. In contrast to the patch experiments they are always symmetric and do not display a voltage offset.

Allyl isothiocyanate (AITC) activates nonselective cation currents in human cardiac fibroblasts: possible involvement of TRPA1

The effects of allyl isothiocyanate (AITC), transient receptor potential ankyrin 1 (TRPA1) agonist, on cultured human cardiac fibroblasts were examined by measuring intracellular Ca2+ concentration [Ca2+]i and whole-cell voltage clamp techniques. AITC (200 μM) increased Ca2+ entry in the presence of [Ca2+]i. Ruthenium red (RR) (30 μM), and La3+ (0.5 mM), a general cation channel blocker, inhibited AITC-induced Ca2+ entry. Under the patch pipette filled with Cs+- and EGTA-solution, AITC induced the current of a reversal potential (Er) of approximately +0 mV. When extracellular Na+ ion was changed by NMDG+, the inward current activated by AITC was markedly reduced. La3+ and RR inhibited the AITC-induced current. The conventional RT-PCR analysis, Western blot, and immunocytochemical studies showed TRPA1 mRNA and protein expression. The present study shows the first evidence for functional Ca2+-permeable nonselective cation currents induced by AITC, possibly via TRPA1 in human cardiac fibroblast.

Single or Double Patch-Clamp Recordings In Ex Vivo Slice Preparation: Functional Connectivity, Synapse Dynamics, and Optogenetics.

Patch-clamp recordings are the method of choice to define cell-type specific electrophysiological properties of single neurons and the synaptic connectivity between pairs of connected neurons in brain slices. In combination with optogenetic tools, patch-clamp recordings allow for the investigation of long-range afferent connectivity from identified distant brain areas. Here we describe the necessary equipment to carry out patch clamp recordings, surgical methods for dissection and preparation of horizontal brain slices containing the hippocampus, and a step-by-step guide for establishing patch clamp recordings in the whole-cell configuration. We provide protocols for single neuron stimulation via the patch pipette and for photostimulation experiments that activate axon terminals expressing light sensitive ion channels.

Voltage-dependent calcium channel β subunit-derived peptides reduce excitatory neurotransmission and arterial blood pressure

AimsVoltage-dependent calcium channels (VDCCs) play an important role in various physiological functions in the nervous system and the cardiovascular system. In L-, N-, P/Q-, and R-type VDCCs, β subunit assists the channels for membrane targeting and modulates channel properties. In this study, we investigated whether an inhibition of the β subunit binding to α subunit, the pore-forming main subunit of VDCCs, have any effect on channel activation and physiological functions. Main methodsPeptides derived from the specific regions of β subunit that bind to the α-interaction domain in I-II linker of α subunit were manufactured, presuming that the peptides interrupt α-β subunit interaction in the channel complex. Then, they were tested on voltage-activated Ca2+ currents recorded in acutely isolated trigeminal ganglion (TG) neurons, excitatory postsynaptic currents (EPSCs) in the spinal dorsal horn neurons, and arterial blood pressure (BP) recorded from the rat femoral artery. Key findingsWhen applied internally through patch pipettes, the peptides decreased the peak amplitudes of the voltage-activated Ca2+ currents. After fusing with HIV transactivator of transcription (TAT) sequence to penetrate cell membrane, the peptides significantly decreased the peak amplitudes of Ca2+ currents and the peak amplitudes of EPSCs upon the external application through bath solution. Furthermore, the TAT-fused peptides dose dependently reduced the rat BP when administered intravenously. SignificanceThese data suggest that an interruption of α-β subunit association in VDCC complex inhibits channel activation, thereby reducing VDCC-mediated physiological functions such as excitatory neurotransmission and arterial BP.

Phosphatidylinositol-4,5-Biphosphate (PI(4,5)P2) Is Required for Rapid Endocytosis in Chromaffin Cells.

Objective Phosphoinositides play a regulatory role in clathrin-mediated endocytosis. However, their involvement in clathrin-independent endocytosis termed rapid endocytosis (RE), which is the mode of vesicle recycling during neurotransmitter release by transient fusion (known as kiss-and-run), has not been investigated. Here, we used patch-clamp recording of whole-cell membrane capacitance in adrenal chromaffin cells (ACC) to monitor changes of RE kinetics in response to pharmacological alteration of phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) level by phenylarsine oxide (PAO) or antibody against phosphatidylinositol 4-kinase (AbPI4K). Results We found that PAO and AbPI4K significantly abrogated RE kinetics. Infusion of PI(4,5)P2 through the patch pipette potentiated RE kinetics and reversed PAO- and AbPI4K-induced blockade of RE. Similarly, the application of the bifunctional thiol dithiothreitol (DTT) to PAO-treated cells completely prevented the inhibitory effect of PAO on RE. These findings indicate that PI(4,5)P2 is implicated in the signaling (mechanistic) process of RE in ACC.

How Many Angels Can Dance on the Head of a Patch Pipette? Understanding Neuronal Hyperexcitability in Angelman Syndrome.

Potassium Channel Dysfunction in Human Neuronal Models of Angelman Syndrome Sun AX, Yuan Q, Fukuda M, Yu W, Yan H, Lim GGY, Nai MH, D’Agostino GA, Tran H-D, Itahana Y, Wang D, Lokman H, Itahana K, Lim SWL, Tang J, Chang YY, Zhang M, Cook SA, Rackham OJL, Lim CT, Tan EK, Ng HH, Lim KL, Jiang Y-H, Je HS. Science. 2019;366(6472):1486-1492. doi: 10.1126/science.aav5386Disruptions in the ubiquitin protein ligase E3A (UBE3A) gene cause Angelman syndrome (AS). Whereas AS model mice have associated synaptic dysfunction and altered plasticity with abnormal behavior, whether similar or other mechanisms contribute to network hyperactivity and epilepsy susceptibility in AS patients remains unclear. Using human neurons and brain organoids, we demonstrate that UBE3A suppresses neuronal hyperexcitability via ubiquitin-mediated degradation of calcium and voltage-dependent big potassium (BK) channels. We provide evidence that augmented BK channel activity manifests as increased intrinsic excitability in individual neurons and subsequent network synchronization. Big potassium antagonists normalized neuronal excitability in both human and mouse neurons and ameliorated seizure susceptibility in an AS mouse model. Our findings suggest that BK channelopathy underlies epilepsy in AS and support the use of human cells to model human developmental diseases.

The Phosphodiesterase Inhibitor IBMX Blocks the Potassium Channel THIK-1 from the Extracellular Side

The two-pore domain potassium channel (K2P-channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA, we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording, we found that THIK-1 currents were inhibited by application of IBMX with an IC50 of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK-1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K2P-channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA.

The Phosphodiesterase Inhibitor IBMX Blocks the Potassium Channel THIK-1 from the Extracellular Side.

The two-pore domain potassium channel (K2P-channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA, we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording, we found that THIK-1 currents were inhibited by application of IBMX with an IC50 of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK-1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K2P-channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA.

Connexin43 and connexin50 channels exhibit different permeability to the second messenger inositol triphosphate

Gap junction channels made of different connexins have distinct permeability to second messengers, which could affect many cell processes, including lens epithelial cell division. Here, we have compared the permeability of IP3 and Ca2+ through channels made from two connexins, Cx43 and Cx50, that are highly expressed in vertebrate lens epithelial cells. Solute transfer was measured while simultaneously monitoring junctional conductance via dual whole-cell/perforated patch clamp. HeLa cells expressing Cx43 or Cx50 were loaded with Fluo-8, and IP3 or Ca2+ were delivered via patch pipette to one cell of a pair, or to a monolayer while fluorescence intensity changes were recorded. Cx43 channels were permeable to IP3 and Ca2+. Conversely, Cx50 channels were impermeable to IP3, while exhibiting high permeation of Ca2+. Reduced Cx50 permeability to IP3 could play a role in regulating cell division and homeostasis in the lens.

A fine‐tuned interaction between the ER and mitochondria regulates Ca2+‐dependent after hyperpolarizations in vasopressin supraoptic neurons

It is known that vasopressin magnocellular neurons become hyperexcitable in cardiovascular diseases such as heart failure. This hyperexcitability is mediated by shifts in both synaptic and intrinsic mechanisms. One key intrinsic mechanism is the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium‐dependent K+ current (IsAHP) most notable for its role in spike frequency adaptation. While features of the sAHP and its effects on firing are well established, a comprehensive understanding of the underlying mechanisms are still unknown. Previous work demonstrated that sAHP activation requires Ca2+ entry via voltage‐gated Ca2+ channels (VGCCs). However, whether in VP neurons Ca2+ entry via these channels directly activates the sAHP, or alternatively, whether it stimulates Ca2+‐induced Ca2+ release from an internal source such as endoplasmic reticulum (ER) or mitochondria remains unknown. Combining patch clamp electrophysiology with simultaneous Ca2+ imaging in transgenic eGFP‐VP rats, we investigated the role of ER and mitochondrial in shaping the sAHP. We measured sAHPs in voltage clamp by evoking a train of action potentials and measuring the resulting outward current. Pre‐incubation of slices with thapsigargin (3μM), an inhibitor of the sarco/ER Ca2+ ATPase inhibited IsAHPs significantly (96%, p< 0.001) while leaving Ca2+ transients unaffected (p > 0.05). This demonstrates that ER Ca2+ release is necessary for sAHP activation. Conversely, blockade of mitochondrial Ca2+ release via blocking mitochondrial Ca2+ exchangers with TPP (20μM) failed to inhibit the IsAHP (p > 0.05). We next evaluated the role of mitochondrial Ca2+ uptake with bath application of the mitochondrial uncoupler CCCP (1μM) or the mitochondrial Ca2+ uniporter (MCU) blocker Ru360 (20μM) in the patch pipette. These manipulations significantly prolonged the decay time constant of both the IsAHP (p< 0.01) and the underlying Ca2+ transient (p< 0.05), demonstrating that the MCU regulates spatiotemporal dynamics of the sAHP. Finally, combining thapsigargin with Ru360 in the pipette showed an initial block of the sAHP, followed by an enhancement over 15 minutes as Ru360 dialyzed into the cell. These results support the ER (but not mitochondria) as a major source of Ca2+ activating the sAHP, as well as a role for the MCU in regulating the magnitude and time course of the sAHP. Taken together, our studies support a fine‐tuned interaction between VGCCs, ER, and mitochondria in modulating the magnitude and time course of the sAHP in VP neurons. Future studies warrant investigation into whether changes in these interactions contribute to altered VP neuronal activity in cardiovascular disease states.Support or Funding InformationR01HL090948

Do Action Potentials in Vasopressin Magnocellular Neurons Back‐propagate from Soma to Dendrites to Evoke Neuropeptide Dendritic Release?

In addition to releasing their peptidergic cargo into the systemic circulation from their axons in the posterior pituitary, magnocellular neurosecretory vasopressin (VP) and oxytocin (OT) neurons in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei also release these neuropeptides from their dendrites. Dendritic release of VP and OT play important functional roles, including recruitment and coordination of neurohumoral responses to an osmotic stimulation. Dendritic release of neuropeptides involves Ca2+‐dependent exocytosis, which in other neuronal types, including substantia nigra dopaminergic neurons, has been shown to be triggered by backpropagating action potentials (bAPs). However, it is at present unknown whether action potentials in VP neurons can back‐propagate from soma to dendrites, and whether they participate in dendritic release of VP. To directly test this, we performed simultaneous dual patch‐clamp electrophysiological recordings from somata (whole‐cell, current‐clamp) and dendrites (loose patch, voltage clamp) in identified VP neurons of eGFP‐VP rats. Various degrees and patters of somatic firing activity were evoked by direct current injection through the somatic patch pipette. Surprisingly, we found evidence for bAPs only in a very small proportion of recorded VP neurons (~20%, 91/435 recordings). When present, bAPs appeared as fast inward deflections that occurred within less than 1 millisecond from the evoked somatic AP. bAPs displayed high fidelity, even at relatively high somatic firing frequencies (up to 10 Hz), with almost no propagation failures observed within the measured distance from the soma. The presence or absence of bAPs was not dependent on neuronal input resistance, dendritic access resistance or dendritic distance from the soma (15–100 μM) Similarly, neither the number, frequency nor pattern (continuous or bursting) of somatic action potential had an influence on the incidence or properties of bAPs (p> 0.5 in all cases). Blockade of K+ channels with 4AP (5 mM n=11) or TEA (30 mM, n=7) did not enabled somatic action potentials to back‐propagate to the patched dendrite. Ongoing morphometric analysis of patched neurons aim at determining whether back‐propagation of action potential correlates with specific morphological features of these neurons. Taken together, our results indicate that despite being highly electrotonically compact, the vast majority of neurosecretory VP neurons do not show evidence for bAPS. These findings raise important questions regarding mechanisms underlying activity‐dependent dendritic release of neuropeptides in hypothalamic neurons.Support or Funding InformationNIH R01NS094640 to JES

Ca2+/Calmodulin Binding to STIM1 Hydrophobic Residues Facilitates Slow Ca2+-Dependent Inactivation of the Orai1 Channel.

Store-operated Ca2+ entry (SOCE) through plasma membrane Ca2+ channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca2+ store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca2+-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca2+ release-activated Ca2+ current (ICRAC). A major regulatory mechanism that prevents deleterious Ca2+ overload is the slow Ca2+-dependent inactivation (SCDI) of ICRAC. Several studies have suggested a role of Ca2+/calmodulin (Ca2+/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca2+/CaM-mediated SCDI of ICRAC is as yet unclear. The Ca2+/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca2+ influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca2+/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology. Here we identified residues Leu374/Val375 (H1) and Leu390/Phe391 (H2) within SOAR that serve as hydrophobic anchor sites for Ca2+/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca2+/CaM binding. Single residue mutations of Phe391 to less hydrophobic residues significantly diminished SOCE and ICRAC, independent of Ca2+/CaM. Hence, the role of H2 residues in Ca2+/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca2+/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of ICRAC caused by Ca2+/CaM, proving a direct role of STIM1 in coordinating SCDI. Taken together, we propose a mechanistic model, wherein binding of Ca2+/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca2+/CaM are functionally coordinated to control ICRAC.

Caloric restriction triggers morphofunctional remodeling of astrocytes and enhances synaptic plasticity in the mouse hippocampus

Calorie-restricted (CR) diet has multiple beneficial effects on brain function. Here we report morphological and functional changes in hippocampal astrocytes in 3-months-old mice subjected to 1 month of the diet. Whole-cell patch-clamp recordings were performed in the CA1 stratum (str.) radiatum astrocytes of hippocampal slices. The cells were also loaded with fluorescent dye through the patch pipette. CR did not affect the number of astrocytic branches but increased the volume fraction (VF) of distal perisynaptic astrocytic leaflets. The astrocyte growth did not lead to a decrease in the cell input resistance, which may be attributed to a decrease in astrocyte coupling through the gap junctions. Western blotting revealed a decrease in the expression of Cx43 but not Cx30. Immunocytochemical analysis demonstrated a decrease in the density and size of Cx43 clusters. Cx30 cluster density did not change, while their size increased in the vicinity of astrocytic soma. CR shortened K+ and glutamate transporter currents in astrocytes in response to 5 × 50 Hz Schaffer collateral stimulation. However, no change in the expression of astrocytic glutamate transporter 1 (GLT-1) was observed, while the level of glutamine synthetase (GS) decreased. These findings suggest that enhanced enwrapping of synapses by the astrocytic leaflets reduces glutamate and K+ spillover. Reduced spillover led to a decreased contribution of extrasynaptic N2B containing N-methyl-D-aspartate receptors (NMDARs) to the tail of burst-induced EPSCs. The magnitude of long-term potentiation (LTP) in the glutamatergic CA3–CA1 synapses was significantly enhanced after CR. This enhancement was abolished by N2B-NMDARs antagonist. Our findings suggest that astrocytic morphofunctional remodeling is responsible for enhanced synaptic plasticity, which provides a basis for improved learning and memory reported after CR.

Robust RNA-Seq of aRNA-amplified single cell material collected by patch clamp

Most single cell RNA sequencing protocols start with single cells dispersed from intact tissue. High-throughput processing of the separated cells is enabled using microfluidics platforms. However, dissociation of tissue results in loss of information about cell location and morphology and potentially alters the transcriptome. An alternative approach for collecting RNA from single cells is to re-purpose the electrophysiological technique of patch clamp recording. A hollow patch pipette is attached to individual cells, enabling the recording of electrical activity, after which the cytoplasm may be extracted for single cell RNA-Seq (“Patch-Seq”). Since the tissue is not disaggregated, the location of cells is readily determined, and the morphology of the cells is maintained, making possible the correlation of single cell transcriptomes with cell location, morphology and electrophysiology. Recent Patch-Seq studies utilizes PCR amplification to increase amount of nucleic acid material to the level required for current sequencing technologies. PCR is prone to create biased libraries – especially with the extremely high degrees of exponential amplification required for single cell amounts of RNA. We compared a PCR-based approach with linear amplifications and demonstrate that aRNA amplification (in vitro transcription, IVT) is more sensitive and robust for single cell RNA collected by a patch clamp pipette.