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W Stühmer

Publications and source records attributed to W Stühmer.

At least 73 records · Page 4Linked to original sources
PubMed1989

Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain.

Cloning and sequencing of cDNAs isolated from a rat cortex cDNA library reveals that a gene family encodes several highly homologous K+ channel forming (RCK) proteins. Functional characterization of the channels expressed in Xenopus laevis oocytes following microinjection of in vitro transcribed RCK-specific RNAs shows that each of the RCK proteins forms K+ channels that differ greatly in both their functional and pharmacological properties. This suggests that the molecular basis for the diversity of voltage-gated K+ channels in mammalian brain is based, at least partly, on the expression of several RCK proteins by a family of genes and their assembly to homooligomeric K+ channels with different functional properties.

4-Aminopyridine↗
PubMed1989

Quantal charge redistributions accompanying the structural transitions of sodium channels.

Asymmetric displacement currents, Ig, associated with the gating of nerve sodium channels have been recorded in cell-attached macropatches of Xenopus laevis oocytes injected with exogenous mRNA coding for rat-brain-II sodium channels. The Ig properties were found to be similar to those of gating currents previously observed in native nerve preparations. Ig fluctuations were measured in order to ascertain the discreteness of the conformational changes which precede the channel opening. The auto-correlation of the fluctuations is consistent with a shot-like character of the elementary Ig contributions. The variance of the fluctuations indicates that most of the gating-charge movement that accompanies the activation of a single sodium channel occurs in 2 to 3 brief packets, each carrying an equivalent of about 2.3 electron charges.

Animals↗
PubMed1989

Intracellular magnesium blocks sodium outward currents in a voltage- and dose-dependent manner.

Tail currents through Na+ channels have been measured in inside-out patches from Xenopus laevis oocytes injected with cDNA-derived mRNA coding for the rat brain type II Na+ channel. It is shown that intracellular Mg2+ blocks outward currents in a voltage- and dose-dependent manner with a half blocking concentration between 3 and 4 mM at 0 mV and a voltage dependence of e-fold per 49 mV.

Animals↗
PubMed1988

Potassium channels expressed from rat brain cDNA have delayed rectifier properties.

Injection into Xenopus oocytes of RNA synthesized in vitro using the rat brain cDNA RCK1 as a template or nuclear injection of the cDNA results in the expression of functional potassium channels. These channels exhibit properties similar to those of the non-inactivating delayed rectifier channel found in mammalian neurons and other excitable cells.

4-Aminopyridine↗
PubMed1988

Slow sodium channel inactivation in mammalian muscle: a possible role in regulating excitability.

Sodium currents were recorded in rat fast and slow twitch muscle fibers. Changes in the membrane potential around the resting potential produced slow changes in the sodium current amplitude due to alterations of the slow inactivation process that was increased by steady depolarization and removed by prolonged hyperpolarization. In contrast, classical fast inactivation was not operative around the resting potential, and depolarizations of greater than 20 mV were required to close half of the channels by fast inactivation. Because slow inactivation is operative around the resting potential of mammalian muscle fibers, it may partially explain why small depolarizations, such as those that occur in some patients with periodic paralysis, can reduce excitability.

Animals↗
PubMed1987

Patch clamp characterization of sodium channels expressed from rat brain cDNA.

Sodium currents, INa, were recorded from Xenopus laevis oocytes which had been injected with mRNA synthesized by in vitro transcription of the rat brain sodium channel II cDNA (Noda et al. 1986 a, b). Patch pipettes were used to apply depolarizing voltage steps and to record macroscopic sodium currents of between 50 and 750 pA from cell-attached patches of the oocyte membrane. With a combination of whole-cell and patch clamp recording, the properties of the implanted sodium channels could be studied in detail. They were analyzed according to the model of Hodgkin and Huxley (1952 a) assuming three activation gates. The activation of the sodium currents is characterized by an equilibrium potential of -29 mV and an apparent gating charge of 8.7 e0. At -64 mV half of the sodium currents were inactivated. From single-channel current recordings, an elementary sodium channel conductance of 19 pS and an average open time of 0.43 ms were obtained at -32 mV membrane potential and 16 degrees C. The single-channel and activation properties of rat brain sodium channel II are therefore comparable to those found in peripheral nerve and skeletal muscle, but inactivation occurs at less negative potentials. This could be a specific property of the brain sodium channels and may underlie the maintained inward sodium currents reported in brain neurones (French and Gage 1985).

Animals↗
PubMed1987

Single acetylcholine receptor channel currents recorded at high hydrostatic pressures.

A technique for performing patch-clamp experiments under high hydrostatic (oil) pressure is described. The method allows the transfer of whole cell or membrane patches in a recording configuration into a pressure vessel, where pressure can be increased up to 60 MPa (approximately equal to 600 bar). We have studied in this way the pressure dependence of single acetylcholine receptor channels in excised "outside-out" membrane patches from cultured rat muscle cells. In the range of 0.1 to 60 MPa the open channel conductance in 140 mM NaCl solutions did not vary by more than 2%, which implies that the translocation of sodium ions through the channel pore does not involve steps with significant activation volumes. At high acetylcholine concentrations (20 microM) bursts of single-channel activity allowed measurements of the mean open and mean closed times of the channel. Pressurization to 40 MPa increased both mean open and mean closed times giving apparent activation volumes of about 59 and 139 A3, respectively. This implies a net volume increase of 80 A3, associated with the transition from the agonist-free state to the open state of the channel, which may be partially associated with the agonist-binding step. All the observed pressure effects were reversible. The activation volumes for the gating of acetylcholine receptor channels are comparable to those of sodium and potassium channels in the squid giant axon, suggesting that there is some basic common mechanism in the operation of ion-channel proteins.

Animals↗
PubMed1987

Effects of hydrostatic pressure on membrane processes. Sodium channels, calcium channels, and exocytosis.

A patch-clamp study under high hydrostatic pressure was performed by transferring cells or membrane patches into a pressure vessel (Heinemann, S. H., W. Stühmer, and F. Conti, 1987, Proceedings of the National Academy of Sciences, 84:3229-3233). Whole-cell Na currents as well as Ca currents were measured at pressures up to 40 MPa (approximately 400 atm; 1 MPa = 9.87 atm) in bovine adrenal chromaffin cells. Ca currents were found to be independent of pressure within experimental resolution. The mean amplitude and the gating kinetics of Na currents were affected by less than 20% at 10 MPa. This lack of a pronounced effect is surprising since the high-pressure nervous syndrome (HPNS), a disorder at high pressures known to result from impaired nervous transmission, manifests itself at pressures as low as 5 MPa. The results show that ion channels involved in transmission cannot be implicated in HPNS. However, when exocytosis was studied at high pressure by monitoring the cell capacitance (Neher, E., and A. Marty, 1982, Proceedings of the National Academy of Sciences, 79:6712-6716), more drastic effects were seen. The degranulation evoked by dialyzing the cell with 1 microM free Ca2+ could be slowed by a factor of 2 by application of 10 MPa. The same effect was observed for the degranulation of rat peritoneal mast cells stimulated with 40 microM of the GTP analogue GTP-gamma-S. According to these results, the process of exocytosis is the most likely site at which hydrostatic pressure can act to produce nervous disorders. Furthermore, we demonstrate that pressure can be a useful tool in the investigation of other cellular responses, since we were able to separate different steps occurring during exocytosis owing to their different activation volumes.

Adrenal Medulla↗
PubMed1987

Slow sodium channel inactivation in rat fast-twitch muscle.

1. Voltage-clamp Na+ currents (INa) were measured in rat fast-twitch fibres using the loose-patch-clamp technique. Changes in the conditioning membrane potential produced slow changes in the peak INa elicited by short test depolarizations, due to a slow inactivation process. 2. Inactivation was increased by application of steady depolarizing potentials and was reversed by steady hyperpolarizations. These changes in peak INa could be well fitted by single-exponential functions with time constants in the range of 1-4 min. 3. The steady-state values of the maximum peak INa at any potential could be well fitted by a function identical to the one describing the fast inactivation process. This gave a potential of -108 mV at which 50% of the channels were closed due to slow inactivation. 4. The maximum peak current densities obtained with the slow inactivation fully removed were as large as 20 mA cm-2.

Animals↗
PubMed1987

Comparison between slow sodium channel inactivation in rat slow- and fast-twitch muscle.

1. Voltage-clamp Na+ currents (INa) were studied in rat soleus slow-twitch muscle fibres at about 18 degrees C using the loose-patch-clamp technique. The maximum inward current density was produced by depolarizations to about -19 mV. 2. Fast inactivation was studied utilizing 20 ms conditioning potentials. INa was reduced by 50% with conditioning potentials to about -70 mV. 3. Changes in the conditioning membrane potential produced slow changes in the peak INa due to a slow inactivation process. INa was reduced by 50% at about -86 mV due to slow inactivation. 4. The mean maximum inward INa when slow inactivation was fully removed was 6.83 mA cm-2. 5. Due to the slow inactivation process, slow-twitch fibres were less susceptible to reduction in INa than fast-twitch fibres.

Animals↗
PubMed1986

Mobility of voltage-dependent ion channels and lectin receptors in the sarcolemma of frog skeletal muscle.

The mobility of lectin receptors and of two types of ion channels was studied in skeletal muscles of the frog Rana temporaria. Lectin receptors were labeled with fluorescent derivatives of succinyl-concanavalin A (Con A) or wheat germ agglutinin (WGA), and their mobility was measured by fluorescence recovery after photobleaching. Of the receptors for WGA, approximately 53% were free to diffuse in the plane of the membrane, with an average diffusion coefficient as found in other preparations (D = 6.4 X 10(-11) cm2/s). Con A receptors were not measurably mobile. The mobility of voltage-dependent Na and K (delayed rectifier) channels was investigated with the loose-patch clamp method, coupled with through-the-pipette photodestruction of channels by ultraviolet (UV) light. Na channels were not measurably mobile (D less than or equal to 10(-12) cm2/s). With K channels, photodestruction was followed by a small but consistent recovery of K current, which suggested that some K channels diffused in the plane of the membrane. Our results with K currents are best fit if 25% of the K channels diffuse with D = 5 X 10(-11) cm2/s, with the remainder being immobile. For both Na and K channels, photodestruction by UV was most effective at a wavelength of approximately 289 nm. At this wavelength, the energy density required for an e-fold reduction in the number of functional channels was 0.40 J/cm2 for Na channels and 0.94 J/cm2 for K channels. Irradiation at this wavelength and dose did not measurably diminish the mobility of WGA receptors; hence, the immobility of Na and most K channels is not due to UV irradiation. It is concluded that mobile and immobile membrane proteins coexist in the sarcolemma of frog skeletal muscle, and that voltage-dependent Na and K channels are singled out for immobilization.

Animals↗
PubMed1984

Voltage-dependent sodium channels in an invertebrate striated muscle.

Striated skeletal muscles from the planktonic arrowworm Sagitta elegans (phylum Chaetognatha) were voltage-clamped. The muscles displayed classical voltage-dependent sodium channels that (i) showed peak transient currents when the membrane was depolarized 90 millivolts from rest, (ii) opened rapidly with peak currents flowing within 0.4 milliseconds at 4 degrees C, (iii) showed voltage-dependent inactivation with 50 percent inactivation at +25 millivolts from rest, and (iv) were blocked by 500 nanomolar tetrodotoxin.

Action Potentials↗
PubMed1984

Pressure dependence of sodium gating currents in the squid giant axon.

Asymmetric displacement currents, Ig, were measured in squid axons at different hydrostatic pressures, P, up to 60 MPa. Potassium and sodium currents were abolished by intracellular Cs+ and TEA+, by extracellular Tetrodotoxin (TTX), and by Na+ substitution with Tris+. The time course of Ig became progressively slower with increasing pressure, and the amplitude decreased. With appropriate scaling in time and amplitude, Ig records at any given P could be made to superimpose very well with those obtained at atmospheric pressure. The same scaling factors yielded a good superposition of all records obtained for voltage steps to membrane potentials in the range -30 to +42 mV. The ratio between the amplitude and time factors was larger than unity and increased with P, indicating a progressive decrease (up to 35% at 60 MPa) of the total charge displaced, Q, with no significant change in its voltage dependence. The time-scaling factor increased exponentially with P, as expected if all the steps involved in the opening of a sodium channel, and producing a major charge redistribution, have the same activation volume, delta V not equal to g approximately 17 cm3/mol. This value is roughly one-half of that characterizing the pressure dependence of sodium current activation, suggesting that some late, rate-limiting step in the opening of sodium channels has a large activation volume without being accompanied by an easily detected charge movement. Part of the decrease of Q with pressure could be attributed to an increase in sodium inactivation. However, we cannot exclude the possibility that there is a reversible reduction in the number of fast activating sodium channels, similar to the phenomenon that has been reported to occur at low temperatures (Matteson and Armstrong 1982).

Animals↗
PubMed1983

Lateral distribution of sodium and potassium channels in frog skeletal muscle: measurements with a patch-clamp technique.

We describe a method for recording Na+ and K+ currents (INa and IK) from small, voltage-clamped patches of sarcolemma by means of fire-polished glass micropipettes of 7-15 microns tip diameter. Recordings can be made successively from many areas of one fibre. On a given fibre, the amplitudes of INa and IK varied from point to point. Maximum Na+ current densities varied up to three-fold over distances of 10-30 microns, typically between 4 and 12 mA/cm2. K+ currents showed somewhat less lateral variation. Local densities of INa and IK showed no correlation. Apparently the density of Na+ (and, to a lesser extent, K+) channels varies laterally. A contour map of Na+ channel density is constructed for a 20 microns X 90 microns section of sarcolemma. Based on the steepness of lateral gradients in channel density and the estimated survival time of a Na+ channel, it is calculated that at least half of the Na+ channels have a lateral diffusion coefficient of less than 2 X 10(-12) cm2/s. This is three orders of magnitudes less than expected from their molecular size, and suggests that these channels are anchored in the sarcolemma.

Animals↗
PubMed1983

Slow changes in currents through sodium channels in frog muscle membrane.

We used a patch clamp to measure Na currents across 10-15 micron diameter circular patches of frog and rat skeletal muscle membrane. We tested for electrophoretic mobility of Na channels, by applying steady lateral fields (of the order of 10 mV micron-1) across the wall of the patch pipette. Application of steady negative potentials to the inside of the pipette resulted in a fall in the number of functional Na channels in the patch. This fall took several minutes to complete and was reversible. It was assayed by applying suitable depolarizations at approximately 11 sec intervals. When a steady lateral field was applied in the absence of changes in membrane potential of the patch, the loss of Na current was virtually abolished. Thus it was not due to electrophoretic movement of channels, but instead to depolarization of the sarcolemma. Evidently, a very slow inactivation of Na conductance operates in skeletal muscle. In frog muscle, the rate constants for loss and recovery of Na current were about 0.1 min-1 (17 degrees C) at resting potential. Rate constants were higher at more positive and at more negative membrane potentials. Current amplitude was reduced to 0.5 at about -76 mV. Roughly similar results were found in rat omohyoid muscle. A further inactivation mechanism, whose rate was intermediate between conventional fast inactivation and the very slow process described here, was present also in both rat and frog muscle. In frog muscle, lateral fields do not alter the potential dependence of fast inactivation. Either the surface charge due to membrane lipids does not influence inactivation or the lipids immediately surrounding the Na channel are restricted in their mobility.

Animals↗
PubMed1982

Effect of glucocorticoid treatment on the excitability of rat skeletal muscle.

Dexamethasone treatment in the rat produced depolarization of extensor digitorum longus (EDL) muscle fibers but not soleus (SOL) fibers studied in vitro at 23 degrees C. The depolarization of EDL fibers was most prominent after 1 day of treatment (treated -77.5 +/- 1.1 mV, control -87.2 +/- 0.8 mV; mean +/- S.E.), and was associated with elevation of the action potential threshold and reduction of the action potential overshoot. In vivo, or in vitro in chloride-free solution, the resting potential and action potential threshold and overshoot of EDL fibers from glucocorticoid-treated and control rats were similar. Sodium currents were studied with a patch voltage clamp. Glucocorticoid treatment did not alter the voltage dependence of sodium channel activation or inactivation in fast twitch muscle fibers. Maximal inward currents occurred at about -29 mV and half-maximal inward currents at about -50 mV. Sodium channels were half inactivated at about -71 mV. Glucocorticoid treatment did not alter the sarcolemmal resistance or capacitance. We conclude that glucocorticoid treatment does not produce muscle weakness or atrophy by altering the excitability of muscle fibers.

Action Potentials↗