The development of severe epilepsy and cognitive decline in children carrying 5 of the 7 studied KCNQ2 mutations can be related to a dominant-negative reduction of the resulting potassium current at subthreshold membrane potentials. Other factors such as genetic modifiers have to be postulated for the remaining 2 mutations. Retigabine or similar drugs may be used as a personalized therapy for this severe disease.

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Bidirectional transport of molecules between nucleus and cytoplasm through the nuclear pore complexes (NPCs) spanning the nuclear envelope plays a fundamental role in cell function and metabolism. Nuclear import of macromolecules is a two-step process involving initial recognition of targeting signals, docking to the pore and energy-driven translocation. ATP depletion inhibits the translocation step. The mechanism of translocation itself and the conformational changes of the NPC components that occur during macromolecular transport, are still unclear. The present study investigates the effect of ATP on nuclear pore conformation in isolated nuclear envelopes from Xenopus laevis oocytes using the atomic force microscope. All experiments were conducted in a saline solution mimicking the cytosol using unfixed nuclear envelopes. ATP (1 mM) was added during the scanning procedure and the resultant conformational changes of the NPCs were directly monitored. Images of the same nuclear pores recorded before and during ATP exposure revealed dramatic conformational changes of NPCs subsequent to the addition of ATP. The height of the pores protruding from the cytoplasmic surface of the nuclear envelope visibly increased while the diameter of the pore opening decreased. The observed changes occurred within minutes and were transient. The slow-hydrolyzing ATP analogue, ATP-gamma-S, in equimolar concentrations did not exert any effects. The ATP-induced shape change could represent a nuclear pore "contraction."

Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2͞3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 ؎ 0.34 S͞cm 2 ). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 ؋ 10 6 NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically ''open'' state with a mean single NPC electrical conductance of 1.7 ؎ 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism.nucleus ͉ electrophysiology N uclear envelopes (NEs) of Xenopus laevis oocytes have been the favored preparation for evaluation of nuclear transport and for studies of nuclear pore complexes (NPCs) structure and function, but only limited data are available on the electrophysiological properties of this important barrier, mainly because of technical limitations.Electrophysiological evaluation of NE started in the early sixties. Using two microelectrodes impaling the nucleus of a Drosophila salivary gland cell, Loewenstein and his coworkers (1) measured the electrical conductance of the NE. The conductance was much smaller than they had expected. At that time, NPCs were thought to be open gaps in the nuclear membrane with a diameter of at least 40 nm. From their electrical conductance measurements, Loewenstein and coworkers concluded that a protein structure must be present in these gaps that restricts the opening to less than 10 nm, resulting in an electrical conductance of about 1 nS per nuclear pore (2). This observation came very close to the view of the nuclear pore that has emerged as a result of much later studies, in which the diffusion of differently sized molecules was measured. According to this view, the NPC forms an aqueous channel of 8-12 nm in diameter and 40-50 nm in length (3). From this data, a NPC electrical conductance of 1-2 nS can be calculated (4).In ...

Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel. We tested whether a gradient of intracellular Ca2+-concentration ([Ca2+]i) underlies the horizontal polarization of K+ channel activity. [Ca2+]i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca2+]i indicated that a horizontal gradient exists, with [Ca2+]i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca2+]i in the cell body were found to be 135 +/- 34 and 405 +/- 59 nml/l, respectively, whereas they were 79 +/- 18 and 307 +/- 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K+ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca2+]i to test whether its maintenance is due to an uneven distribution of Ca2+ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La3+ alone or together with charybdotoxin, a specific blocker of Ca2+-sensitive K+ channels, slowed migration to 47 +/- 10% and 9 +/- 5% of control, respectively. Local blockade of Ca2+ influx into the cell body and the lamellipodium with la3+ was followed by a decrease of [Ca2+]i at both cell poles. This points to Ca2+ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn2+ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca2+]i. We hypothesize that the polarized distribution of intracellular Ca2+ stores contributes significantly to the generation of a gradient of [Ca2+]i.

Migration of transformed renal epithelial cells (transformed Madin-Darby canine kidney cells, MDCK-F cells) relies on the activity of a Ca(2+)-sensitive K+ channel (IK channel) that is more active at the rear end of these cells. We have postulated that intermittent IK channel activity induces local cell shrinkage at the rear end of migrating MDCK-F cells and thereby supports the cytoskeletal mechanisms of migration. However, due to the complex morphology of MDCK-F cells we have not yet been able to measure volume changes directly. The aim of the present study was to devise a new technique employing atomic force microscopy (AFM) to measure the volume of MDCK-F cells in their physiological environment and to demonstrate its dependence on IK channel activity. The spatial (x, y' and z) co-ordinates of each pixel of the three-dimensional image of MDCK-F cells allow calculation of the volume of the column "underneath" a given pixel. Thus, total cell volume is the sum of all pixel-defined columns. The mean volume of 17 MDCK-F cells was 2500+/-300 fl. Blockade of the IK channel with the specific inhibitor charybdotoxin (CTX) increased cell volume by 17+/-4%; activation of IK by elevating the intracellular [Ca2+] with the Ca2+ ionophore ionomycin decreased cell volume by 19+/-3%. Subtraction images (experimental minus control) reveal that swelling and shrinkage occur predominantly at the rear end of MDCK-F cells. In summary, our experiments show that AFM allows the measurement not only of total cell volume of living cells in their physiological environment but also the tracing of local effects induced by the polarized distribution of K+ channel activity.

Blockade of the cardiac ion channel coded by human ether-à-gogo-related gene (hERG) can lead to cardiac arrhythmia, which has become a major concern in drug discovery and development. Automated electrophysiological patch clamp allows assessment of hERG channel effects early in drug development to aid medicinal chemistry programs and has become routine in pharmaceutical companies. However, a number of potential sources of errors in setting up hERG channel assays by automated patch clamp can lead to misinterpretation of data or false effects being reported. This article describes protocols for automated electrophysiology screening of compound effects on the hERG channel current. Protocol details and the translation of criteria known from manual patch clamp experiments to automated patch clamp experiments to achieve good quality data are emphasized. Typical pitfalls and artifacts that may lead to misinterpretation of data are discussed. While this article focuses on hERG channel recordings using the QPatch (Sophion A/S, Copenhagen, Denmark) technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms.

Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed (inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing oocytes contained about 200 proteins per micron 2. Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mM isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per micron 2 while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 400 proteins per micron 2 while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular level. We propose that CFTR causes clustering of plasma membrane proteins.