Klaus Benndorf

Expression pattern of neuronal and skeletal muscle voltage-gated Na+ channels in the developing mouse heart

In the mammalian heart, a variety of voltage‐gated Na+ channel transcripts and proteins have been detected. However, little quantitative information is available on the abundance of each transcript during development, or the contribution of TTX‐sensitive Na+ channels to the cardiac sodium current (INa). Using competitive and real‐time RT‐PCR we investigated the transcription of six Na+ channels (Nav1.1–Nav1.6) and the β1 subunit during mouse heart development. Nav1.5 was predominantly expressed in the adult heart, whereas the splice variant Nav1.5a was the major Na+ channel isoform in embryonic hearts. The TTX‐resistant Na+ channel transcripts (Nav1.5 and Nav1.5a) increased 1.7‐fold during postnatal development. Transcripts encoding TTX‐sensitive Na+ channels (Nav1.1–Nav1.4) and the β1 subunit gradually increased up to fourfold from postnatal day (P)1 to P126, while the Nav1.6 transcript level remained low and constant over the same period. In adults, TTX‐sensitive channel mRNA accounted for 30–40% of the channel pool in whole‐heart preparations (Nav1.3 > Nav1.4 > Nav1.2 ≫ Nav1.1 ∼ Nav1.6), and 16% in mRNA from isolated cardiomyocytes (Nav1.4 > Nav1.3 > Nav1.2 > Nav1.1 > Nav1.6). Confocal immunofluorescence on ventricular myocytes suggested that Nav1.1 and Nav1.2 were localized at the intercalated disks and in the t tubules. Nav1.3 labelling predominantly produced a diffuse but strong intracellular signal. Nav1.6 fluorescence was detected only along the Z lines. Electrophysiological recordings showed that TTX‐sensitive and TTX‐resistant Na+ channels, respectively, accounted for 8% and 92% of the INa in adult ventricular cardiomyocytes. Our data suggest that neuronal and skeletal muscle Na+ channels contribute to the action potential of cardiomyocytes in the adult mammalian heart.

Relating ligand binding to activation gating in CNGA2 channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory signal transduction in photoreceptors and olfactory cells. Structurally, CNG channels are heterotetramers composed of either two or three homologue subunits. Although it is well established that activation is a cooperative process of these subunits, it remains unknown whether the cooperativity is generated by the ligand binding, the gating, or both, and how the subunits interact. In this study, the action of homotetrameric olfactory-type CNGA2 channels was studied in inside-out membrane patches by simultaneously determining channel activation and ligand binding, using the fluorescent cGMP analogue 8-DY547-cGMP as the ligand. At concentrations of 8-DY547-cGMP < 1 μM, steady-state binding was larger than steady-state activation, whereas at higher concentrations it was smaller, generating a crossover of the steady-state relationships. Global analysis of these relationships together with multiple activation time courses following cGMP jumps showed that four ligands bind to the channels and that there is significant interaction between the binding sites. Among the binding steps, the second is most critical for channel opening: its association constant is three orders of magnitude smaller than the others and it triggers a switch from a mostly closed to a maximally open state. These results contribute to unravelling the role of the subunits in the cooperative mechanism of CNGA2 channel activation and could be of general relevance for the action of other ion channels and receptors.

FRET between cardiac Na + channel subunits measured with a confocal microscope and a streak camera

When and where proteins associate is a central question in many biomolecular studies. Förster resonance energy transfer (FRET) measurements can be used to address this question when the interacting proteins are labeled with appropriate donor and acceptor fluorophores. We describe an improved method to determine FRET efficiency that uses a mode-locked laser, a confocal microscope and a streak camera. We applied this method to study the association of α and β1 subunits of the human cardiac sodium channel. The subunits were tagged with the cyan and yellow variants of the green fluorescent protein (GFP) and expressed in human embryonic kidney (HEK293) cells. Pronounced FRET between the channel subunits in the endoplasmic reticulum (ER) suggested that the subunits associate before they reach the plasma membrane. The described method allows simultaneous measurement of donor and acceptor fluorescence decays and provides an intrinsically validated estimate of FRET efficiency.

Anoxia induces time‐independent K+ current through KATP channels in isolated heart cells of the guinea‐pig.

1. Isolated ventricular heart cells of the guinea‐pig were exposed to anoxia (PO2 < 0.1 Torr) which induced a time‐independent K+ current. This current was studied with the patch clamp technique in the whole‐cell and cell‐attached configuration. 2. The latency until anoxia‐induced changes of whole‐cell current developed was distributed exponentially (mean 10.5 min; n = 41). The current was abolished within 2‐4 s of reoxygenation. 3. The reversal potential of the anoxia‐induced change of whole‐cell current at 5.4 and 15 mM [K+]o was ‐82 and ‐61 mV, respectively. 4. Analysis of current noise in whole‐cell current during the phase of the slow development of the anoxia‐induced current yielded a slope conductance of unitary currents of 8.1 pS (5.4 mM [K+]o) which is far below the 30 pS of KATP channels in inside‐out patches with no Na+ and Mg2+ in the bath. 5. Reduced unitary current induced by anoxia was recorded in single‐channel measurements with 10.4 mM‐K+ in the pipette. 6. Using 150 mM‐K+ in the pipette, anoxia caused unitary inward currents with a slope conductance of 83 pS. The open probability of the channels (P(o)) reached maximum values between 0.6 and 0.95. The channels closed within 1‐3 s of reoxygenation. 7. At voltages between ‐85 and ‐45 mV and maximum P(o), open time histograms were dominated by a fast exponential (tau 01 = 0.55 +/‐ 0.21 ms, mean +/‐ S.D.) and one or two slow exponentials. 8. Voltage ramp experiments showed that single‐channel currents were slightly rectifying in the inward direction. 9. Glibenclamide (1 microM) reversibly blocked the anoxia‐induced whole‐cell and single‐channel currents. 10. It is concluded that during anoxia it is only KATP channels which open by a sufficient decrease of submembrane ATP levels.

FRET Measurements by TCSPC Laser Scanning Microscopy

We use a two-photon laser scanning microscope with a new Time-Correlated Single Photon Counting (TCSPC) imaging technique to obtain combined intensity-lifetime images for FRET measurements in living cells. Single photon pulses from a photomultiplier and signals from the scanning head are used to record the three-dimensional photon density over the time- and image coordinates. Double exponential decay analysis delivers the lifetime components of the quenched and the unquenched molecules in all pixels of the image. We use the ratio of the intensity coefficients of the fast and slow decay component to create images that show the size of the FRET effects in different parts of the cell.

Localization of functional calcitonin gene-related peptide binding sites in a subpopulation of cultured dorsal root ganglion neurons

In this study we investigated whether cultured dorsal root ganglion (DRG) neurons from the adult rat express binding sites for calcitonin gene-related peptide (CGRP). These were identified on fixed cells by using CGRP labeled at the N-terminal site with 1.4-nm gold particles. After 1 day in culture, about 20% of small to medium-sized DRG neurons showed CGRP-gold binding. Binding of CGRP-gold was dose-dependently reduced by coadministration of CGRP. The calcium imaging technique in living cells revealed that the bath administration of CGRP evoked an increase of the intracellular calcium in up to 30% of the DRG neurons tested. Both depletion of intracellular calcium stores by thapsigargin or using a calcium-free medium blocked the CGRP-mediated increase of cytosolic calcium in most neurons. Thus intracellular and extracellular sources of calcium are relevant for the CGRP response. Using the whole-cell patch-clamp technique, about 30% of the neurons were found to exhibit an inward current and a depolarization upon administration of CGRP close to the neurons. Immunocytochemical double-labeling techniques showed that most of the CGRP-gold binding sites were expressed in unmyelinated (neurofilament 200-negative) DRG neurons. Most of the neurons with CGRP-gold binding sites also expressed the tyrosine kinase A receptor, and all of them showed CGRP-like immunoreactivity. This study shows, therefore, that a subpopulation of unmyelinated, peptidergic primary afferent neurons express CGRP binding sites that can be activated by CGRP in an excitatory direction. The binding sites may serve as autoreceptors because all of these neurons also synthesize CGRP. The activation of CGRP binding sites may sensitize primary afferent neurons and influence the release of mediators.

Interdependence of Receptor Activation and Ligand Binding in HCN2 Pacemaker Channels

HCN pacemaker channels are tetramers mediating rhythmicity in neuronal and cardiac cells. The activity of these channels is controlled by both membrane voltage and the ligand cAMP, binding to each of the four channel subunits. The molecular mechanism underlying channel activation and the relationship between the two activation stimuli are still unknown. Using patch-clamp fluorometry and a fluorescent cAMP analog, we show that full ligand-induced activation appears already with only two ligands bound to the tetrameric channel. Kinetic analysis of channel activation and ligand binding suggests direct interaction between the voltage sensor and the cyclic nucleotide-binding domain, bypassing the pore. By exploiting the duality of activation in HCN2 channels by voltage and ligand binding, we quantify the increase of the binding affinity and overall free energy for binding upon channel activation, proving thus the principle of reciprocity between ligand binding and conformational change in a receptor protein.

Functional interaction between K(ATP) channels and the Na(+)‐K(+) pump in metabolically inhibited heart cells of the guinea‐pig.

1. Transmembrane current through ATP‐regulated K(+) channels (IK(ATP)) was measured in ventricular heart cells of the guinea‐pig in the whole‐cell and cell‐attached patch configurations under conditions of metabolic poisoning with the mitochondrial uncoupler 2,4‐dinitrophenol (DNP). 2. Maintained exposure of the cells to DNP resulted in a transient appearance of whole‐cell IK(ATP) When IK(ATP) had reached several nanoamps, blocking the forward‐running Na(+)‐K(+) pump with 0.5 mM strophanthidin decreased IK(ATP) after a delay. The time course of this decrease could be described by a single exponential function, which yielded a time constant(T)of 4.51+/‐ 1.89 s (n=8). 3. Hyperpolarization from 0 mV to ‐100 or ‐150 mV for 2 s caused IK(ATP) (measured at 0 mV) to decrease by 34.2 +/‐ 14.1 % (n = 8) and 37.6 +/‐ 9.4% (n = 8), respectively. After the hyperpolarizing pulse, IK(ATP) returned to its higher initial level within a couple of seconds. 4. Driving the pump backwards by removing the extracellular K(+) ions caused the permanent disappearance of DNP‐induced IK(ATP). 5. Application of 0.5 mM strophanthidin in the absence of external K(+) ions induced a transient increase in IK(ATP), as did washing out the glycoside (n = 5). 6. When pump action was inhibited by using Na(+), K(+)‐free Tyrode solution (see Methods) in the bath, strophanthidin did not have a comparable direct effect on IK(ATP). 7. In cell‐attached patches, strophanthidin applied via the bath caused a reduction in IK(ATP) with a similar time course to that in whole‐cell experiments. This suggests that the interaction between the pump molecules and the K(ATP) channels is not restricted to closely neighbouring molecules. 8. The data support the hypothesis that [ATP] at the cytosolic face of the membrane may drop to practically zero, thereby passing an ‘ATP window’ in which the channels first open and then close, and that the submembrane [ATP] is readily controlled by the cytosolic [ATP].

BK channel blockers inhibit potassium-induced proliferation of human astrocytoma cells.

The functional role of BK channels, which are consistently expressed in glioma cells, is not clear. Here we show that the BK channels are regularly active in human 1321N1 astrocytoma cells at physiological membrane potentials. The proliferation of the cells at the physiological external [K+] of 5 mM is compared with that at the elevated external [K+] of 20 mM, simulating the situation in rapidly growing, necrotic tumours in vivo. High extracellular [K+] in the range 10–30 mM significantly increases the proliferation of 1321N1 cells. This K+ induced proliferation can be completely abolished by applying the specific BK channel blockers iberiotoxin (IBTX) or 1 mM tetraethylammonium (TEA). Neither blocker has any effect on cell growth at 5 mM [K+]e. These findings indicate a particular role of BK channels in astrocytoma cell proliferation.

Functional Expression of GFP-linked Human Heart Sodium Channel (hH1) and Subcellular Localization of the a Subunit in HEK293 Cells and Dog Cardiac Myocytes

Recent evidence suggests that biosynthesis of the human heart Na+ channel (hH1) protein is rapidly modulated by sympathetic interventions. However, data regarding the intracellular processing of hH1 in vivo are lacking. In this study we sought to establish a model that would allow us to study the subcellular localization of hH1 protein. Such a model could eventually help us to better understand the trafficking of hH1 in vivo and its potential role in cardiac conduction. We labeled the C-terminus of hH1 with the green fluorescent protein (GFP) and compared the expression of this construct (hH1-GFP) and hH1 in transfected HEK293 cells. Fusion of GFP to hH1 did not alter its electrophysiological properties. Confocal microscopy revealed that hH1-GFP was highly expressed in intracellular membrane structures. Immuno-electronmicrographs showed that transfection of hH1-GFP and hH1 induced proliferation of three types of endoplasmic reticulum (ER) membranes to accommodate the heterologously expressed proteins. Labeling with specific markers for the ER and the Golgi apparatus indicated that the intracellular channels are almost exclusively retained within the ER. Immunocytochemical labeling of the Na+ channel in dog cardiomyocytes showed strong fluorescence in the perinuclear region of the cells, a result consistent with our findings in HEK293 cells. We propose that the ER may serve as a reservoir for the cardiac Na+ channels and that the transport from the ER to the Golgi apparatus is among the rate-limiting steps for sarcolemmal expression of Na+ channels.