Search PubMed⌕ Search

Biomedical subjects

W Almers

Publications and source records attributed to W Almers.

At least 73 records · Page 4Linked to original sources
PubMed1985

The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading.

The Ca concentration ([Ca2+]i) in single rat peritoneal mast cells was measured by means of the new fluorescent Ca-indicator dye fura-2. Dye-loaded cells were made to degranulate with either antigen or compound 48/80. In cells loaded with extracellularly applied, membrane-permeant fura-2 ester, degranulation was accompanied by a permanent loss of 40-60% of the fluorescence, but comparison of fluorescence at different wavelengths indicated no or only small changes in [Ca2+]i. When cells were loaded by microinjection of the impermeant potassium salt of the dye, degranulation resulted in no permanent loss of fluorescence, but instead was preceded by transient fluorescence changes that indicate a rapid, large and transient increase in [Ca2+]i. We suggest that ester-loaded fura-2 accumulates to a significant degree in the secretory granules and is lost from the cell during exocytosis.

Animals↗
PubMed1985

Slow calcium and potassium currents in frog skeletal muscle: their relationship and pharmacologic properties.

Slow Ca and K currents across frog skeletal muscle membrane were recorded with the Vaseline gap voltage clamp in order to investigate block by divalent cations and various organic compounds. Cd2+, Ni2+, Co2+, Mn2+, Mg2+ all block Ca currents, as do barbiturates, D-600 and nifedipine. Local anesthetics also block Ca currents, with the impermeant quaternary lidocaine derivative, OX-314, being more than an order of magnitude less potent than its permeant parent compound. Surprisingly, all agents that blocked Ca currents also blocked the slow K currents. To explain this pharmacologic parallel, one could suggest that K current is activated by Ca2+ appearing in the myoplasm due to the combination of Ca current and release from internal stores. While possibly correct for intact fibres, this hypothesis appears not to apply in our case where the myoplasm contained the Ca chelator EGTA at high concentration. Instead, K currents seem to be activated by a decrease in external [Ca2+]. In the transverse tubules, Ca current is known to cause [Ca2+] to decline to submicromolar concentrations, and evidence is presented that K currents are activated by Ca depletion from a restricted extracellular space. It is suggested that K currents flow through Ca channels that have become capable of passing monovalent cations after the tubules have become depleted of Ca2+.

Anesthetics, Local↗
PubMed1985

The Ca channel in skeletal muscle is a large pore.

The permeability of Ca channels to various foreign cations has been investigated in the absence of external Ca2+. All physiological metal cations are clearly permeant, including Mg2+. The large organic cation n-butylamine+ is sparingly permeant or impermeant, but its larger derivative 1,4-diaminobutane2+ is highly permeant. Among the cations of the methylated ammonium series, permeability diminishes in a graded fashion as ion size increases. Tetramethylammonium, the largest cation found to be permeant, has a diameter of about 6 A; hence, the aqueous pore of the Ca channel at its narrowest point can be no smaller. That the pore is so large strengthens our view that, under physiologic conditions, the high selectivity of Ca channels is due to selective binding of Ca2+ rather than to rejection of other cations by, for example, a sieving mechanism.

Animals↗
PubMed1984

An improved loose patch voltage clamp method using concentric pipettes.

A method for noninvasive voltage-clamp recording from large cells is described. A firepolished pipette having two concentric barrels is pushed against the cell membrane, thereby electrically isolating a circular patch subdivided into an inner and an annular outer region. Both regions are held isopotential, but current is collected from the inner region only. The method electrically simulates a high resistance seal between pipette and cell membrane, allowing accurate and rapid voltage-clamp recording under conditions where the seal resistances actually obtained are low (near 1 M omega). This is useful in applications where one wishes to avoid enzymatic treatment. We provide details of electrode construction and voltage-clamp electronics, and present results obtained from frog skeletal muscle and leech neurons. For sodium channels of frog muscle, extensive data were previously obtained with other methods. There is good agreement between the earlier results and the measurements presented here.

Animals↗
PubMed1984

Voltage clamp of rat and human skeletal muscle: measurements with an improved loose-patch technique.

Intact fibres of human intercostal and rat omohyoid muscles were studied at 23 degree C with a loose-patch voltage-clamp technique that employed two concentric micropipettes to electrically isolate small-diameter (10-15 microns) patches of sarcolemma. This method allows investigation of membrane excitability under highly physiological conditions. Step depolarizations to 0 mV elicited sodium inward currents that reached peak values of up to 20 mA/cm2 within 250 microseconds, and then declined. In human muscle, the reversal potential (ENa) was approximately 40 mV, and maximal conductances (GNa) ranged from 44 to 360 mS/cm2. In rat muscle, ENa was 42 mV and GNa ranged from 100 to 250 mS/cm2. Sodium channels in rat and human muscle were indistinguishable in most aspects of their kinetic behaviour and voltage dependence. Outward potassium currents were small by comparison (usually less than 2 mA/cm2) and saturated at positive potentials. The maximum potassium conductance (GK) ranged from 0 to 19 mS/cm2 (human) and from 4 to 12 mS/cm2 (rat muscle).

Action Potentials↗
PubMed1984

A non-selective cation conductance in frog muscle membrane blocked by micromolar external calcium ions.

Membrane currents were recorded from voltage-clamped, EGTA-loaded muscle fibres under conditions where currents through ordinary Na+, K+ and Cl- channels were prevented by drugs or by absence of permeant ions (K+, and Cl-). At 10 mM-external [Ca2+], substitution of Na+ for the large and presumably impermeant organic cations tetramethyl- (TMA+) or tetraethylammonium (TEA+) failed to increase peak inward current. Hence the Ca2+ channel was not significantly permeable to Na+ under these conditions. When external [Ca2+] was reduced to levels below 1 microM in the presence of external Na+, step depolarizations to negative potentials produced tetrodotoxin-resistant inward currents. At -20 mV, they rose to a peak of 30-200 microA/cm2 within 150 ms and declined thereafter. Ca2+ and several other divalent cations reversibly blocked this inward current. The sequence of blocking potencies was Ca2+ greater than Sr2+ greater than or equal to Co2+ greater than Mn2+ congruent to Cd2+ greater than Ni2+ congruent to Mg2+. Large inward currents may be carried by Li+, Na+, K+, Rb+ and Cs+ but not by TMA+ and TEA+. The effect of external Ca2+ ([Ca2+]o) was explored over a 10(8)-fold range in concentrations. Na+ was present at a fixed concentration. When [Ca2+]o was gradually increased from 10(-10) to 10(-2) M, inward current first diminished 10-fold, reached a minimum at [Ca2+]o = 60 microM and then increased again as [Ca2+]o was increased further and Ca2+ itself became a current carrier. Block of inward current at [Ca2+]o less than 10(-5) M could be described by binding of a single Ca2+ to a site, with a dissociation constant of the order of 0.7 microM at -20 mV.

Action Potentials↗
PubMed1984

Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore.

Voltage-clamp studies were carried out to compare currents through Ca2+ channels (ICa) with Na+ currents (Ins) through a non-selective cation conductance blocked by micromolar concentrations of external Ca2+. The gating of both currents was found to have similar time and voltage dependence. The amplitudes of ICa and Ins varied widely, but Ins was always large in fibres with large ICa, and small in fibres with small ICa. Both ICa and Ins were blocked by the specific Ca2+ channel blocker nifedipine, with half-blockage concentrations that were virtually identical (KD = 0.9 microM for ICa and 0.7 microM for Ins). ICa and Ins were also equally sensitive to block by diltiazem (KD = 80 microM). These parallels between Ins and ICa are most easily explained if Ins flows through Ca2+ channels. Apparently, Ca2+ channels bear high-affinity Ca2+-binding sites, and are highly permeable to monovalent cations when Ca2+ is absent. Ba2+ currents (IBa) and ICa were measured in external solutions containing mixtures of Ba2+ and Ca2+. IBa is blocked by Ca2+, as is Ins. Adding Ba2+ to Ca2+ produces only small or no increases in current, as if Ba2+ is only sparingly permeant when Ca2+ is present. Membrane currents in Ba2+/Ca2+ mixtures show anomalous mole-fraction behaviour, suggesting that Ca2+ channels are single-file, multi-ion pores. Complex current transients are observed under maintained depolarizations in Na+/Ca2+ and Ba2+/Ca2+ mixtures. They suggest that in ion mixtures, Ca2+ channels transport Ca2+ in preference to Na+ and Ba2+. Hence Ca2+ channels are selective for Ca2+, even though current amplitudes suggest that the Na+ or Ba2+ permeabilities in the absence of Ca2+ are as high as, or higher than, the Ca2+ permeability. We conclude that the selective permeability of Ca2+ channels depends on the presence of Ca2+. In model calculations, our observations are explained as a consequence of Ca2+ channels being single-file pores. It is proposed that Ca2+ channels derive much of their ion selectivity from high-affinity Ca2+ binding sites located in an otherwise unselective aqueous pore.

Action Potentials↗
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↗
PubMed1982

Photobleaching through glass micropipettes: sodium channels without lateral mobility in the sarcolemma of frog skeletal muscle.

Sodium currents were recorded from frog skeletal muscle by using fire-polished micropipettes to electrically isolate and voltage clamp a small patch of sarcolemma. Sodium current amplitude served as an assay for the number of functional sodium channels in the patch. With the pipette as a light guide, these channels were irradiated with ultraviolet (UV) light directed through a quartz fiber into the back end of the pipette. The UV light emerging from the pipette tip caused localized destruction of the sodium channels in the patch, reducing sodium current 3- to 5-fold during a 30-90 s irradiation. If sodium channels could diffuse laterally in the membrane, current from the patch should recover with time as fresh channels enter from neighboring areas. No such recovery was observed during observation for 1 hr after irradiation. Our results set an upper limit of 10(-12) cm2/s for the diffusion coefficient--1/1000th that of rhodopsin, a membrane protein in the cell membrane of retinal rods. It is suggested that sodium channels are anchored in the sarcolemma.

Ion Channels↗
PubMed1981

Slow calcium and potassium currents across frog muscle membrane: measurements with a vaseline-gap technique.

1. A vaseline-gap voltage-clamp technique was used to record slow Ca2+ and K+ currents from frog skeletal muscle fibres loaded with the Ca2+ chelator EGTA. 2. K+ currents were increased when Mg2+ replaced external Ca2+, and they were abolished when internal K+ was replaced by tetraethylammonium (TEA+). Ca2+ currents could be studied in isolation in fibres loaded with (TEA)2EGTA. 3. Under maintained depolarization, Ca2+ currents slowly increase (half-time of 35 msec or more at 25 mV) and then decline to a steady value. Decline under repolarization is rapid (half-time of 6-7 msec) and complete. During an action potential, the Ca2+ influx through this system is probably less than the influx observed with tracers. 4. Ba2+, Sr2+, Ca2+, Mn2+ and Mg2+ can carry current across the membrane; Ni2+ and Co2+ cannot. Ca2+ currents are weakly blocked by external Mg2+.

Animals↗
PubMed1981

Calcium depletion in frog muscle tubules: the decline of calcium current under maintained depolarization.

1. Ca2+ currents in frog skeletal muscle fibres were studied with a voltage-clamp technique. Under membrane depolarization maintained for several seconds, Ca2+ current was found to decline with time constants of 0.2-2 sec when [Ca2+]o = 10 mM. 2. Ca2+ currents are diminished by nifedipine, D-600, tetracaine and Ni2+. 3. When peak current is diminished by making the membrane potential positive, by block with drugs or by substituting the relatively less permeant Mn2+ for Ca2+ then the rate of decline is diminished also. When peak current is increased by recording at relatively negative membrane potentials or by substituting for Ca2+ the more permeant ions Ba2+ or Sr2+, then the rate of decline is increased in proportion. Evidently, the size of the current determines the rate of decline. 4. Decline of current is greatly slowed in isotonic Ca2+ saline or when the [Ca2+]o is buffered by the organic anion malate. These findings indicate that the decline of current arises from Ca2+ depletion in an extracellular compartment, most probably the transverse tubules. On this basis, an analysis of Ca2+ current decline and recovery leads to the following conclusions. 5. Ca2+ current flows almost entirely across the membranes of the transverse tubules. 6. After allowing for the tortuosity of the tubular network, the apparent diffusion coefficient for Ca2+ in the transverse tubules is about 2.6 X 10(-6) cm2/sec, three times less than the diffusion coefficient for K+ in the transverse tubules and about three times less than the diffusion coefficient for Ca2+ in free solution. 7. The transverse tubule lumen does not appear to have a large Ca2+-buffering capacity in the millimolar range. At [Ca2+]o = 10 mM, the tubule lumen binds less than 0.6 dissociable Ca2+ ions for every free ion.

Animals↗
PubMed1980

Survival of K+ permeability and gating currents in squid axons perfused with K+-free media.

K+ currents were recorded in squid axons internally perfused with impermeant electrolyte. Total absence of permeant ions inside and out leads to an irreversible loss of potassium conductance with a time constant of approximately 11 min at 8 degrees C. Potassium channels can be protected against this effect by external K+, Cs+, NH4+, and Rb+ at concentrations of 100-440 mM. These experiments suggest that a K+ channel is normally occupied by one or more small cations, and becomes nonfunctional when these cations are removed. A large charge movement said to be related to K+ channel gating in frog skeletal muscle is absent in squid giant axons. However, deliberate destruction of K+ conductance by removal of permeant cations is accompanied by measurable loss in asymmetric charge movement. This missing charge component is large enough to contain a contribution from K+ gating charge movements of more than five elementary charges per channel.

Animals↗
PubMed1980

Potassium concentration changes in the transverse tubules of vertebrate skeletal muscle.

Vertebrate skeletal muscle fibers have evolved a network of narrow tube-like invaginations of the cell membrane. This "transverse tubular system" (TTS) provides a pathway for radial impulse propation from the cell surface to the interior. As a consequence of electrical activity in the TTS, impulses are followed by "early" and "late" afterdepolarizations. The late afterdepolarization is now believed to be due mainly to K+-accumulation in the TTS. Excessively large afterdepolarizations of this type may be the cause of the "myotonic discharge" observed in mammals suffering from pathologically low muscle membrane permeability to chloride. Potassium concentration changes in the transverse tubular system can also be induced artificially under voltage-champ conditions. Analysis of K+-depletion under voltage clamp allows conclusions about the localization of K+-permeability as well as speed of K+-diffusion in the transverse tubules.

Action Potentials↗
PubMed1979

Interactions between quaternary lidocaine, the sodium channel gates, and tetrodotoxin.

A voltage clamp technique was used to study sodium currents and gating currents in squid axons internally perfused with the membrane impermeant sodium channel blocker, QX-314. Block by QX-314 is strongly and reversibly enhanced if a train of depolarizing pulses precedes the measurement. The depolarization-induced block is antagonized by external sodium. This antagonism provides evidence that the blocking site for the drug lies inside the channel. Depolarization-induced block of sodium current by QX-314 is accompanied by nearly twofold reduction in gating charge movement. This reduction does not add to a depolarization-induced immobilization of gating charge normally present and believed to be associated with inactivation of sodium channels. Failure to act additively suggests that both, inactivation and QX-314, affect the same component of gating charge movement. Judged from gating current measurement, a drug-blocked channel is an inactivated channel. In the presence of external tetrodotoxin and internal QX-314, gating charge movement is always half its normal size regardless of conditioning, as it QX-314 is then permanently present in the channel.

Animals↗
PubMed1979

Block of sodium conductance and gating current in squid giant axons poisoned with quaternary strychnine.

Quaternary strychnine blocks sodium channels from the axoplasmic side, probably by insertion into the inner channel mouth. Block is strongly voltage dependent, being more pronounced in depolarized than in resting axons. Using potential steps as a means to modulate the level of block, we investigate strychnine effects on sodium and gating currents at +50 and -50 mV. We analyze our data in terms of the simplest possible model, wherein only an open channel may receive and retain a strychnine molecule. Our main findings are (a) block by strychnine and inactivation resemble each other and (b) block of sodium and gating currents by strychnine happen with closely similar time-courses. Our data support the hypothesis of Armstrong and Bezanilla (1977) wherein an endogenous blocking particle causes inactivation by inserting itself into the inner mouth of the sodium channel. Quaternary strychnine may act as an artificial substitute for the hypothetical endogenous blocking particle. Further, we suggest that at least 90% of the rapid asymmetrical displacement current in squid axons is sodium channel gating current, inasmuch as quaternary strychnine can block 90% of the displacement current simultaneously with sodium current.

Animals↗