The G-protein–gated K+ channel, IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K+ current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4−/− mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4−/− mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4−/− animals. Although the relative extent of heart rate regulation of Girk4−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4−/− animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation

Effect of Cytosolic pH on Inward Currents Reveals Structural Characteristics of the Proton Transport Cycle in the Influenza A Protein M2 in Cell-Free Membrane Patches of Xenopus oocytes.

Transport activity through the mutant D44A of the M2 proton channel from influenza virus A was measured in excised inside-out macro-patches of Xenopus laevis oocytes at cytosolic pH values of 5.5, 7.5 and 8.2. The current-voltage relationships reveal some peculiarities: 1. "Transinhibition", i.e., instead of an increase of unidirectional outward current with increasing cytosolic H+ concentration, a decrease of unidirectional inward current was found. 2. Strong inward rectification. 3. Exponential rise of current with negative potentials. In order to interpret these findings in molecular terms, different kinetic models have been tested. The transinhibition basically results from a strong binding of H+ to a site in the pore, presumably His37. This assumption alone already provides inward rectification and exponential rise of the IV curves. However, it results in poor global fits of the IV curves, i.e., good fits were only obtained for cytosolic pH of 8.2, but not for 7.5. Assuming an additional transport step as e.g. caused by a constriction zone at Val27 resulted in a negligible improvement. In contrast, good global fits for cytosolic pH of 7.5 and 8.2 were immediately obtained with a cyclic model. A "recycling step" implies that the protein undergoes conformational changes (assigned to Trp41 and Val27) during transport which have to be reset before the next proton can be transported. The global fit failed at the low currents at pHcyt = 5.5, as expected from the interference of putative transport of other ions besides H+. Alternatively, a regulatory effect of acidic cytosolic pH may be assumed which strongly modifies the rate constants of the transport cycle

Interfacing Graphene-Based Materials With Neural Cells

The scientific community has witnessed an exponential increase in the applications of graphene and graphene-based materials in a wide range of fields, from engineering to electronics to biotechnologies and biomedical applications. For what concerns neuroscience, the interest raised by these materials is two-fold. On one side, nanosheets made of graphene or graphene derivatives (graphene oxide, or its reduced form) can be used as carriers for drug delivery. Here, an important aspect is to evaluate their toxicity, which strongly depends on flake composition, chemical functionalization and dimensions. On the other side, graphene can be exploited as a substrate for tissue engineering. In this case, conductivity is probably the most relevant amongst the various properties of the different graphene materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation, which holds a great potential in regenerative medicine. In this review, we try to give a comprehensive view of the accomplishments and new challenges of the field, as well as which in our view are the most exciting directions to take in the immediate future. These include the need to engineer multifunctional nanoparticles (NPs) able to cross the blood-brain-barrier to reach neural cells, and to achieve on-demand delivery of specific drugs. We describe the state-of-the-art in the use of graphene materials to engineer three-dimensional scaffolds to drive neuronal growth and regeneration in vivo, and the possibility of using graphene as a component of hybrid composites/multi-layer organic electronics devices. Last but not least, we address the need of an accurate theoretical modeling of the interface between graphene and biological material, by modeling the interaction of graphene with proteins and cell membranes at the nanoscale, and describing the physical mechanism(s) of charge transfer by which the various graphene materials can influence the excitability and physiology of neural cells

The funny current in genetically modified mice

Since its first description in 1979, the hyperpolarization-activated funny current (I-f) has been the object of intensive research aimed at understanding its role in cardiac pacemaker activity and its modulation by the sympathetic and parasympathetic branches of the autonomic nervous system. I-f was described in isolated tissue strips of the rabbit sinoatrial node using the double-electrode voltage-clamp technique. Since then, the rabbit has been the principal animal model for studying pacemaker activity and I-f for more than 20 years. In 2001, the first study describing the electrophysiological properties of mouse sinoatrial pacemaker myocytes and those of I-f was published. It was soon followed by the description of murine myocytes of the atrioventricular node and the Purkinje fibres. The sinoatrial node of genetically modified mice has become a very popular model for studying the mechanisms of cardiac pacemaker activity. This field of research benefits from the impressive advancement of in-vivo exploration tech-niques of physiological parameters, imaging, genetics, and large-scale genomic approaches. The present review discusses the influence of mouse genetic on the most recent knowledge of the funny current's role in the physiology and pathophysiology of cardiac pacemaker activity. Genetically modified mice have provided important insights into the role of I-f in determining intrinsic automaticity in vivo and in myocytes of the conduction system. In addition, gene targeting of f-(HCN) channel isoforms have contributed to elucidating the current's role in the regulation of heart rate by the parasympathetic nervous system. This review is dedicated to Dario DiFrancesco on his retirement. (C) 2021 Published by Elsevier Ltd

Effect of Cytosolic pH on Inward Currents Reveals Structural Characteristics of the Proton Transport Cycle in the Influenza A Protein M2 in Cell-Free Membrane Patches of <i>Xenopus</i> oocytes

<div><p>Transport activity through the mutant D44A of the M2 proton channel from influenza virus A was measured in excised inside-out macro-patches of <i>Xenopus laevis</i> oocytes at cytosolic pH values of 5.5, 7.5 and 8.2. The current-voltage relationships reveal some peculiarities: 1. “Transinhibition”, i.e., instead of an increase of unidirectional outward current with increasing cytosolic H⁺ concentration, a decrease of unidirectional inward current was found. 2. Strong inward rectification. 3. Exponential rise of current with negative potentials. In order to interpret these findings in molecular terms, different kinetic models have been tested. The transinhibition basically results from a strong binding of H⁺ to a site in the pore, presumably His37. This assumption alone already provides inward rectification and exponential rise of the IV curves. However, it results in poor global fits of the IV curves, i.e., good fits were only obtained for cytosolic pH of 8.2, but not for 7.5. Assuming an additional transport step as e.g. caused by a constriction zone at Val27 resulted in a negligible improvement. In contrast, good global fits for cytosolic pH of 7.5 and 8.2 were immediately obtained with a cyclic model. A “recycling step” implies that the protein undergoes conformational changes (assigned to Trp41 and Val27) during transport which have to be reset before the next proton can be transported. The global fit failed at the low currents at pH_cyt = 5.5, as expected from the interference of putative transport of other ions besides H⁺. Alternatively, a regulatory effect of acidic cytosolic pH may be assumed which strongly modifies the rate constants of the transport cycle.</p></div

Calculated IV curves for different external pH of 5.5, 6.5 and 7.5.

<p>The parameters in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107406#pone-0107406-t001" target="_blank">Table 1</a> from the joint fit for pH_cyt = 8.2 with <i>κ_CO</i> modified by pH_ext (Eq. 6) were used to generate the curves; pH_ext is given at the curves. For comparison, triangles show the experimental data of Fig. 6 for pH_cyt = 8.2/pH_ext = 5.5.</p

Properties of the M2D44A transport cycle as calculated from the cyclic model in Fig. 5A.

<p>(A) Occupation probabilities of the three states in the model and their dependence on membrane potential <i>V</i> and cytosolic pH_cyt. (B) Unidirectional currents <i>I_in = −f O k_OB</i> (red) and <i>I_out</i> = <i>f B k_BO</i> (blue, pH 7.5 and 8.2 coincide) and their dependence of membrane potential <i>V</i> and cytosolic pH_cyt. The inset shows the unidirectional outward currents on an expanded scale.</p

Fit of the data of Fig. 4 with the cyclic reaction scheme.

<p>The lines give the fits by means of Eqs. S13–S15 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107406#pone.0107406.s001" target="_blank">file S1</a>. For pH 5.5, two fits are shown: red curve: joint fit with curves for cytosolic pH of 7.5 and 8.2, black curve: without the restriction by the joint fit. This is illustrated by two different “free” fits at pH 5.5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107406#pone-0107406-t001" target="_blank">Table 1</a>, data columns 4 and 5), which show that different sets of rate constants can fit the curve.</p