ANTAGONISM OF SAXITOXIN AND TETRODOTOXIN BLOCK BY INTERNAL MONOVALENT CATIONS IN SQUID AXON

The block of voltage-dependent sodium channels by saxitoxin (STX) and tetrodotoxin (TTX) was investigated in voltage-clamped squid giant axons internally perfused with a variety of permeant monovalent cations. Substitution of internal Na+ by either NH4+ or N2H5+ resulted in a reduction of outward current through sodium channels under control conditions. In contrast, anomalous increases in both inward and outward currents were seen for the same ions if some of the channels were blocked by STX or TTX, suggesting a relief of block by these internal cations. External NH4+ was without effect on the apparent magnitude of toxin block. Likewise, internal inorganic monovalent cations were without effect, suggesting that proton donation by NH4+ might be involved in reducing toxin block. Consistent with this hypothesis, decreases in internal pH mimicked internal perfusion with NH4+ in reducing toxin block. The interaction between internally applied protons and externally applied toxin molecules appears to be competitive, as transient increases in sodium channel current were observed during step increases in intracellular pH in the presence of a fixed STX concentration. In addition to these effects on toxin block, low internal pH produced a voltage-dependent block of sodium channels and enhanced steady-state inactivation. Elevation of external buffer capacity only marginally diminished the modulation of STX block by internal NH4+, suggesting that alkalinization of the periaxonal space and a resultant decrease in the cationic STX concentration during NH4+ perfusion may play only a minor role in the effect. These observations indicate that internal monovalent cations can exert trans-channel influences on external toxin binding sites on sodium channels.

Optogenetic Stimulation Using Anion Channelrhodopsin (GtACR1) Facilitates Termination of Reentrant Arrhythmias With Low Light Energy Requirements: A Computational Study

Optogenetic defibrillation of hearts expressing light-sensitive cation channels (e.g., ChR2) has been proposed as an alternative to conventional electrotherapy. Past modeling work has shown that ChR2 stimulation can depolarize enough myocardium to interrupt arrhythmia, but its efficacy is limited by light attenuation and high energy needs. These shortcomings may be mitigated by using new optogenetic proteins like Guillardia theta Anion Channelrhodopsin (GtACR1), which produces a repolarizing outward current upon illumination. Accordingly, we designed a study to assess the feasibility of GtACR1-based optogenetic arrhythmia termination in human hearts. We conducted electrophysiological simulations in MRI-based atrial or ventricular models (n = 3 each), with pathological remodeling from atrial fibrillation or ischemic cardiomyopathy, respectively. We simulated light sensitization via viral gene delivery of three different opsins (ChR2, red-shifted ChR2, GtACR1) and uniform endocardial illumination at the appropriate wavelengths (blue, red, or green light, respectively). To analyze consistency of arrhythmia termination, we varied pulse timing (three evenly spaced intervals spanning the reentrant cycle) and intensity (atrial: 0.001–1 mW/mm2; ventricular: 0.001–10 mW/mm2). In atrial models, GtACR1 stimulation with 0.005 mW/mm2 green light consistently terminated reentry; this was 10–100x weaker than the threshold levels for ChR2-mediated defibrillation. In ventricular models, defibrillation was observed in 2/3 models for GtACR1 stimulation at 0.005 mW/mm2 (100–200x weaker than ChR2 cases). In the third ventricular model, defibrillation failed in nearly all cases, suggesting that attenuation issues and patient-specific organ/scar geometry may thwart termination in some cases. Across all models, the mechanism of GtACR1-mediated defibrillation was voltage forcing of illuminated tissue toward the modeled channel reversal potential of −40 mV, which made propagation through affected regions impossible. Thus, our findings suggest GtACR1-based optogenetic defibrillation of the human heart may be feasible with ≈2–3 orders of magnitude less energy than ChR2.

5-HT recruits distinct neurocircuits to inhibit hunger-driven and non-hunger-driven feeding

Obesity is primarily a consequence of consuming calories beyond energetic requirements, but underpinning drivers have not been fully defined. 5-Hydroxytryptamine (5-HT) neurons in the dorsal Raphe nucleus (5-HTDRN) regulate different types of feeding behavior, such as eating to cope with hunger or for pleasure. Here, we observed that activation of 5-HTDRN to hypothalamic arcuate nucleus (5-HTDRN → ARH) projections inhibits food intake driven by hunger via actions at ARH 5-HT2C and 5-HT1B receptors, whereas activation of 5-HTDRN to ventral tegmental area (5-HTDRN → VTA) projections inhibits non-hunger-driven feeding via actions at 5-HT2C receptors. Further, hunger-driven feeding gradually activates ARH-projecting 5-HTDRN neurons via inhibiting their responsiveness to inhibitory GABAergic inputs; non-hunger-driven feeding activates VTA-projecting 5-HTDRN neurons through reducing a potassium outward current. Thus, our results support a model whereby parallel circuits modulate feeding behavior either in response to hunger or to hunger-independent cues.

Species-Specific Effects of Cation Channel TRPM4 Small-Molecule Inhibitors

Background: The Transient Receptor Potential Melastatin member 4 (TRPM4) gene encodes a calcium-activated non-selective cation channel expressed in several tissues. Mutations in TRPM4 have been reported in patients with different types of cardiac conduction defects. It is also linked to immune response and cancers, but the associated molecular mechanisms are still unclear. Thus far, 9-phenanthrol is the most common pharmacological compound used to investigate TRPM4 function. We recently identified two promising aryloxyacyl-anthranilic acid compounds (abbreviated CBA and NBA) inhibiting TRPM4. However, all aforementioned compounds were screened using assays expressing human TRPM4, whereas the efficacy of mouse TRPM4 has not been assessed. Mouse models are essential to investigate ion channel physiology and chemical compound efficacy.Aim: In this study, we performed comparative electrophysiology experiments to assess the effect of these TRPM4 inhibitors on human and mouse TRPM4 channels heterologously expressed in TsA-201 cells.Methods and Results: We identified striking species-dependent differences in TRPM4 responses. NBA inhibited both human and mouse TRPM4 currents when applied intracellularly and extracellularly using excised membrane patches. CBA inhibited human TRPM4, both intracellularly and extracellularly. Unexpectedly, the application of CBA had no inhibiting effect on mouse TRPM4 current when perfused on the extracellular side. Instead, its increased mouse TRPM4 current at negative holding potentials. In addition, CBA on the intracellular side altered the outward rectification component of the mouse TRPM4 current. Application of 9-phenanthrol, both intracellularly and extracellularly, inhibited human TRPM4. For mouse TRPM4, 9-phenanthrol perfusion led to opposite effects depending on the site of application. With intracellular 9-phenanthrol, we observed a tendency towards potentiation of mouse TRPM4 outward current at positive holding potentials.Conclusion: Altogether, these results suggest that pharmacological compounds screened using “humanised assays” should be extensively characterised before application in vivo mouse models.

Bidirectional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of −30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that β-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If. Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.

Role of Detrusor PDGFRα+ Cells in Cyclophosphamide-Induced Detrusor Overactivity

Cyclophosphamide (CYP)-induced cystitis is a rodent model that shares many features common to the cystitis occurring in patients, including detrusor overactivity (DO). Platelet-derived growth factor receptor alpha positive (PDGFRα+) cells regulate muscle excitability in murine bladders during filling. PDGFRα+ cells express small conductance Ca2+-activated K+ channels (predominantly SK3) that provide stabilization of membrane potential during filling. We hypothesized that down-regulation of the regulatory functions of PDGFRα+ cells and/or loss of PDGFRα+ cells generates the DO in CYP-treated mice. After CYP treatment, transcripts of Pdgfrα and Kcnn3 were reduced, and PDGFRα and SK3 protein was also reduced in detrusor muscle extracts. The distribution of PDGFRα+ cells was also reduced. Inflammatory markers were increased in CYP-treated detrusor muscles. An SK channel agonist, CyPPA, increased outward current and hyperpolarization in PDGFRα+ cells. This response was significantly depressed in PDGFRα+ cells from CYP-treated bladders. Ex vivo cystometry showed increased transient contractions in CYP-treated bladders, and the sensitivity of these bladders to apamin was reduced, reflecting the reduction in the SK conductance expressed by PDGFRα+ cells. In summary, PDGFRα+ cells were reduced and the SK3 conductance was downregulated in CYP-treated bladders. These changes are consistent with the development of DO after CYP treatment.

Species-specific effects of cation channel TRPM4 small-molecule inhibitors

Background: The Transient Receptor Potential Melastatin member 4 (TRPM4) gene encodes a calcium-activated non-selective cation channel expressed in several tissues. Mutations in TRPM4 have been reported in patients with different types of cardiac conduction defects. It is also linked to immune response and cancers, but the associated molecular mechanisms are still unclear. Thus far, 9-phenanthrol is the most common pharmacological compound used to investigate TRPM4 function. We recently identified two promising aryloxyacyl-anthranilic acid compounds (abbreviated CBA and NBA) inhibiting TRPM4. However, all aforementioned compounds were screened using assays expressing human TRPM4, whereas the efficacy on mouse TRPM4 has not been assessed. Mouse models are essential to investigate ion channel physiology and chemical compound efficacy. Aim: In this study, we performed comparative electrophysiology experiments to assess the effect of these TRPM4 inhibitors on human and mouse TRPM4 channels heterologously expressed in TsA-201 cells. Methods and Results: We identified striking species-dependent differences in TRPM4 responses. NBA inhibited both human and mouse TRPM4 currents when applied intracellularly and extracellularly using excised membrane patches. CBA inhibited human TRPM4, both intracellularly and extracellularly. Unexpectedly, the application of CBA had no inhibiting effect on mouse TRPM4 current when perfused on the extracellular side. Instead, it increased mouse TRPM4 current at negative holding potentials. In addition, CBA on the intracellular side altered the outward rectification component of the mouse TRPM4 current. Application of 9-phenanthrol, both intracellularly and extracellularly, inhibited human TRPM4. For mouse TRPM4, 9-phenanthrol perfusion led to opposite effects depending on the site of application. With intracellular 9-phenanthrol, we observed a tendency towards potentiation of mouse TRPM4 outward current at positive holding potentials. Conclusion: Altogether, these results suggest that pharmacological compounds screened using "humanised assays" should be extensively characterised before application in in vivo mouse models.

Immediate and Delayed Response of Simulated Human Atrial Myocytes to Clinically-Relevant Hypokalemia

Although plasma electrolyte levels are quickly and precisely regulated in the mammalian cardiovascular system, even small transient changes in K+, Na+, Ca2+, and/or Mg2+ can significantly alter physiological responses in the heart, blood vessels, and intrinsic (intracardiac) autonomic nervous system. We have used mathematical models of the human atrial action potential (AP) to explore the electrophysiological mechanisms that underlie changes in resting potential (Vr) and the AP following decreases in plasma K+, [K+]o, that were selected to mimic clinical hypokalemia. Such changes may be associated with arrhythmias and are commonly encountered in patients (i) in therapy for hypertension and heart failure; (ii) undergoing renal dialysis; (iii) with any disease with acid-base imbalance; or (iv) post-operatively. Our study emphasizes clinically-relevant hypokalemic conditions, corresponding to [K+]o reductions of approximately 1.5 mM from the normal value of 4 to 4.5 mM. We show how the resulting electrophysiological responses in human atrial myocytes progress within two distinct time frames:(i) Immediately after [K+]o is reduced, the K+-sensing mechanism of the background inward rectifier current (IK1) responds. Specifically, its highly non-linear current-voltage relationship changes significantly as judged by the voltage dependence of its region of outward current. This rapidly alters, and sometimes even depolarizes, Vr and can also markedly prolong the final repolarization phase of the AP, thus modulating excitability and refractoriness.(ii) A second much slower electrophysiological response (developing 5–10 minutes after [K+]o is reduced) results from alterations in the intracellular electrolyte balance. A progressive shift in intracellular [Na+]i causes a change in the outward electrogenic current generated by the Na+/K+ pump, thereby modifying Vr and AP repolarization and changing the human atrial electrophysiological substrate.In this study, these two effects were investigated quantitatively, using seven published models of the human atrial AP. This highlighted the important role of IK1 rectification when analyzing both the mechanisms by which [K+]o regulates Vr and how the AP waveform may contribute to “trigger” mechanisms within the proarrhythmic substrate. Our simulations complement and extend previous studies aimed at understanding key factors by which decreases in [K+]o can produce effects that are known to promote atrial arrhythmias in human hearts.

Generators of Pressure-Evoked Currents in Vertebrate Outer Retinal Neurons

(1) Background: High-tension glaucoma damages the peripheral vision dominated by rods. How mechanosensitive channels (MSCs) in the outer retina mediate pressure responses is unclear. (2) Methods: Immunocytochemistry, patch clamp, and channel fluorescence were used to study MSCs in salamander photoreceptors. (3) Results: Immunoreactivity of transient receptor potential channel vanilloid 4 (TRPV4) was revealed in the outer plexiform layer, K+ channel TRAAK in the photoreceptor outer segment (OS), and TRPV2 in some rod OS disks. Pressure on the rod inner segment evoked sustained currents of three components: A) the inward current at <−50 mV (Ipi), sensitive to Co2+; B) leak outward current at ³−80 mV (Ipo), sensitive to intracellular Cs+ and ruthenium red; and C) cation current reversed at ~10 mV (Ipc). Hypotonicity induced slow currents like Ipc. Environmental pressure and light increased the FM 1-43-identified open MSCs in the OS membrane, while pressure on the OS with internal Cs+ closed a Ca2+-dependent current reversed at ~0 mV. Rod photocurrents were thermosensitive and affected by MSC blockers. (4) Conclusions: Rods possess depolarizing (TRPV) and hyperpolarizing (K+) MSCs, which mediate mutually compensating currents between −50 mV and 10 mV, serve as an electrical cushion to minimize the impact of ocular mechanical stress.

Capsaicin and Proton Differently Modulate Activation Kinetics of Mouse Transient Receptor Potential Vanilloid-1 Channel Induced by Depolarization

The transient receptor potential vanilloid type 1 (TRPV1) channel is a non-selective cation channel expressed with transient receptor potential ankyrin type 1 (TRPA1) in small and medial size neurons of the dorsal root ganglions and trigeminal ganglions. TRPV1 is activated by capsaicin, thermal stimuli higher than 43°C, mechanical stress, and protons (H+). Although the TRPV1 channel does not have positively charged residues at regular intervals on its transmembrane segments, alterations in membrane potential also affect the state of TRPV1 channel. In the presence of capsaicin, voltage-dependent probability of opening of the TRPV1 channel and its kinetics have been examined, but the characteristics in the low pH remain unclear. To understand the voltage-dependency of the TRPV1 channel activation, we recorded capsaicin- and proton-induced mouse TRPV1 channel currents in a heterologous expression system. Outward current evoked by depolarizing square pulses in the presence of capsaicin or protons was fitted to a two-exponential function with a time-independent component. The voltage-dependent changes in amplitude of the three components displayed shallow curves and the changes in their ratio to the total current display similar tendencies in the presence of capsaicin and under the low pH. However, the fast and slow time constants in the presence of capsaicin were respectively 5- and 8-fold lower than those obtained under low pH conditions. These results suggest that the TRPV1 channel slowly drives the feed-forward cycle of pain sensation, and capsaicin and protons differently modulate the voltage-dependent TRPV1 channel gating.