Search PubMed⌕ Search

Biomedical subjects

K G Beam

Publications and source records attributed to K G Beam.

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

Development alters the expression of calcium currents in chick limb motoneurons.

Calcium current types expressed in vertebrate spinal motoneurons have not been previously resolved. We have resolved three types in chick limb motoneurons identified by retrograde labeling and report that dramatic changes in their expression take place during development in vivo. T-, N-, and L-type calcium currents were distinguished on the basis of kinetics, voltage dependence, and unique pharmacological sensitivities. Developmental changes were characterized by studying motoneurons isolated from embryos at three stages spanning neuromuscular system development. T currents were dominant at the earliest stage. Motoneurons from embryos 2 days older showed much reduced T currents and much increased N and L currents. We suggest that mature motoneurons will be dominated by N- and L-type calcium currents and that T current may serve developmental roles.

Action Potentials↗
PubMed1989

Rescue of excitation-contraction coupling in dysgenic muscle by addition of fibroblasts in vitro.

Muscular dysgenesis (mdg) in mice causes the failure of excitation-contraction (E-C) coupling in skeletal muscle. Cultured dysgenic muscle fails to contract upon depolarization, lacks typical muscle ultrastructure, including normal triads, and lacks functional voltage-dependent slow calcium channels. We show that normal rodent fibroblasts and 3T3 fibroblasts "rescue" dysgenic myotubes, reestablishing contractions (i.e., E-C coupling), normal ultrastructure, and functional slow calcium channels. These results support the finding that the expression of the slow calcium channel is affected in the mdg mutation and that this protein is essential for E-C coupling. Additionally, fibroblast rescue provides a system for examining the mechanisms of heterotypic cellular influence on cell function.

Action Potentials↗
PubMed1989

A novel calcium current in dysgenic skeletal muscle.

The whole-cell patch-clamp technique was used to study voltage-dependent calcium currents in primary cultures of myotubes and in freshly dissociated skeletal muscle from normal and dysgenic mice. In addition to the transient, dihydropyridine (DHP)-insensitive calcium current previously described, a maintained DHP-sensitive calcium current was found in dysgenic skeletal muscle. This current, here termed ICa-dys, is largest in acutely dissociated fetal or neonatal dysgenic muscle and also in dysgenic myotubes grown on a substrate of killed fibroblasts. In dysgenic myotubes grown on untreated plastic culture dishes, ICa-dys is usually so small that it cannot be detected. In addition, ICa-dys is apparently absent from normal skeletal muscle. From a holding potential of -80 mV. ICa-dys becomes apparent for test pulses to approximately -20 mV and peaks at approximately +20 mV. The current activates rapidly (rise time approximately 5 ms at 20 degrees C) and with 10 mM Ca as charge carrier inactivates little or not at all during a 200-ms test pulse. Thus, ICa-dys activates much faster than the slowly activating calcium current of normal skeletal muscle and does not display Ca-dependent inactivation like the cardiac L-type calcium current. Substituting Ba for Ca as the charge carrier doubles the size of ICa-dys without altering its kinetics. ICa-dys is approximately 75% blocked by 100 nM (+)-PN 200-110 and is increased about threefold by 500 nM racemic Bay K 8644. The very high sensitivity of ICa-dys to these DHP compounds distinguishes it from neuronal L-type calcium current and from the calcium currents of normal skeletal muscle. ICa-dys may represent a calcium channel that is normally not expressed in skeletal muscle, or a mutated form of the skeletal muscle slow calcium channel.

Animals↗
PubMed1988

Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA.

Microinjection of an expression plasmid that carries complementary DNA encoding the receptor for dihydropyridine calcium channel blockers of skeletal muscle restores both excitation-contraction coupling and slow calcium current in cultured skeletal muscle cells from mice with muscular dysgenesis. This suggests that the dihydropyridine receptor in the transverse tubule membrane of skeletal muscle functions both as the voltage sensor for excitation-contraction coupling and as the slow calcium channel.

Animals↗
PubMed1988

Apical localization of K+ channels in taste cells provides the basis for sour taste transduction.

Previous studies have shown that mudpuppy taste receptor cells respond to sour taste stimuli (weak acids) with depolarizing receptor potentials or action potentials that are blocked by the K+ channel blocker tetraethylammonium. Voltage-clamp recordings from isolated taste cells indicated that taste receptor cells exhibit a variety of voltage-dependent conductances and that acids reduce a voltage-dependent K+ current. Since taste stimuli are restricted to the apical surface of the intact tongue, only 1-2% of the taste receptor cell surface is exposed to chemical stimuli. Thus, modification of a K+ conductance would be an effective transduction mechanism in receptor cells only if the majority of K+ channels were located on the apical membrane. We have used a combination of "loose-patch" and whole-cell recording methods to map the distribution of voltage-sensitive K+ and Na+ channels on dissociated Necturus maculosus taste cells. We report here that the K+ conductance is approximately equal to 50-fold greater on apical membrane than on basolateral membrane, whereas the Na+ conductance is distributed evenly. The marked nonuniformity of the voltage-sensitive K+ conductance, together with the block of this conductance by sour stimuli, indicates that K+ current modulation is the mechanism of sour taste transduction.

Animals↗
PubMed1988

Calcium currents in embryonic and neonatal mammalian skeletal muscle.

The whole-cell patch-clamp technique was used to study the properties of inward ionic currents found in primary cultures of rat and mouse skeletal myotubes and in freshly dissociated fibers of the flexor digitorum brevis muscle of rats. In each of these cell types, test depolarizations from the holding potential (-80 or -90 mV) elicited three distinct inward currents: a sodium current (INa) and two calcium currents. INa was the dominant inward current: under physiological conditions, the maximum inward INa was estimated to be at least 30-fold larger than either of the calcium currents. The two calcium currents have been termed Ifast and Islow, corresponding to their relative rates of activation. Ifast was activated by test depolarizations to around -40 mV and above, peaked in 10-20 ms, and decayed to baseline in 50-100 ms. Islow was activated by depolarizations to approximately 0 mV and above, peaked in 50-150 ms, and decayed little during a 200-ms test pulse. Ifast was inactivated by brief, moderate depolarizations; for a 1-s change in holding potential, half-inactivation occurred at -55 to -45 mV and complete inactivation occurred at -40 to -30 mV. Similar changes in holding potential had no effect on Islow. Islow was, however, inactivated by brief, strong depolarizations (e.g., 0 mV for 2 s) or maintained, moderate depolarizations (e.g., -40 mV for 60 s). Substitution of barium for calcium had little effect on the magnitude or time course of either Ifast or Islow. The same substitution shifted the activation curve for Islow approximately 10 mV in the hyperpolarizing direction without affecting the activation of Ifast. At low concentrations (50 microM), cadmium preferentially blocked Islow compared with Ifast, while at high concentrations (1 mM), it blocked both Ifast and Islow completely. The dihydropyridine calcium channel antagonist (+)-PN 200-110 (1 microM) caused a nearly complete block of Islow without affecting Ifast. At a holding potential of -80 mV, the half-maximal blocking concentration (K0.5) for the block of Islow by (+)-PN 200-110 was 182 nM. At depolarized holding potentials that inactivated Islow by 35-65%, K0.5 decreased to 5.5 nM.

Animals↗
PubMed1988

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle.

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.

Animals↗
PubMed1986

Na channel distribution in vertebrate skeletal muscle.

The loose patch voltage clamp has been used to map Na current density along the length of snake and rat skeletal muscle fibers. Na currents have been recorded from (a) endplate membrane exposed by removal of the nerve terminal, (b) membrane near the endplate, (c) extrajunctional membrane far from both the endplate and the tendon, and (d) membrane near the tendon. Na current densities recorded directly on the endplate were extremely high, exceeding 400 mA/cm2 in some patches. The membrane adjacent to the endplate has a current density about fivefold lower than that of the endplate, but about fivefold higher than the membrane 100-200 micron from the endplate. Small local variations in Na current density are recorded in extrajunctional membrane. A sharp decrease in Na current density occurs over the last few hundred micrometers from the tendon. We tested the ability of tetrodotoxin to block Na current in regions close to and far from the endplate and found no evidence for toxin-resistant channels in either region. There was also no obvious difference in the kinetics of Na current in the two regions. On the basis of the Na current densities measured with the loose patch clamp, we conclude that Na channels are abundant in the endplate and near-endplate membrane and are sparse close to the tendon. The current density at the endplate is two to three orders of magnitude higher than at the tendon.

Animals↗
PubMed1985

Slow charge movement in mammalian skeletal muscle.

Voltage-dependent charge movements were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Contraction was abolished with hypertonic sucrose. The standard (ON-OFF) protocol for eliciting charge movements was to depolarize the fiber from -90 mV to a variable test potential (V) and then repolarize the fiber to -90 mV. The quantity of charge moved saturated at test potentials of approximately 0 mV. The steady state dependence of the amount of charge that moves as a function of test potential could be well fitted by the Boltzmann relation: Q = Qmax/(1 + exp[-(V - V)/k]), where Qmax is the maximum charge that can be moved, V is the potential at which half the charge moves, and k is a constant. At 15 degrees C, these values were Qmax = 28.5 nC/microF, V = -34.2 mV, and k = 8.7 mV. Qmax, k, and V exhibited little temperature dependence over the range 7-25 degrees C. "Stepped OFF" charge movements were elicited by depolarizing the fiber from -90 mV to a fixed conditioning level that moved nearly all the mobile charge (0 mV), and then repolarizing the fiber to varying test potentials. The sum of the charge that moved when the fiber was depolarized directly from -90 mV to a given test potential and the stepped OFF charge that moved when the fiber was repolarized to the same test potential had at all test potentials a value close to Qmax for that fiber. In nearly all cases, the decay phase of ON, OFF, and stepped OFF charge movements could be well fitted with a single exponential. The time constant, tau decay, for an ON charge movement at a given test potential was comparable to tau decay for a stepped OFF charge movement at the same test potential. Tau decay had a bell-shaped dependence on membrane potential: it was slowest at a potential near V (the midpoint of the steady state charge distribution) and became symmetrically faster on either side of this potential. Raising the temperature from 7 to 15 degrees C caused tau decay to become faster by about the same proportion at all potentials, with a Q10 averaging 2.16. Raising the temperature from 15 to 25 degrees C caused tau decay to become faster at potentials near V, but not at potentials farther away.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

The influence of transverse tubular delays on the kinetics of charge movement in mammalian skeletal muscle.

A model was developed to describe the kinetics of slow, voltage-dependent charge movement in the rat omohyoid muscle. To represent the electrically distributed nature of the transverse tubular system (t-system), we followed an approach similar to that described by Adrian and Peachey (1973 J. Physiol. [Lond.]. 235:103), and approximated the fiber with 12 concentric cylindrical shells. Incorporated into each shell were capacitative and conductive elements that represented the passive electrical properties of the t-system, and an element representing the mobile charge. The charge was assumed to obey a two-state scheme, in which the redistribution of charge is governed by a first-order reaction, and the rate constants linking the two states were assumed to depend on potential according to the constant field expression. The predictions of this "distributed two-state model" were compared with charge movements experimentally measured in individual fibers. For this comparison, first, the passive electrical parameters of the model were adjusted to fit the experimental linear capacity transient. Next, the Boltzmann expression was fitted to the steady state Q vs. V data of the fiber, thereby constraining the voltage dependence of the rate constants, but not their absolute magnitude. The absolute magnitude was determined by fitting the theory to an experimental charge movement at a single test potential, which in turn constrained the fits at all other test potentials. The distributed two-state model well described the rising and falling phases of ON, OFF, and stepped OFF charge movements at temperatures ranging from 3 to 25 degrees C. We thus conclude that tubular delays are sufficient to account for the rounded rising phase of experimental charge movements, and that it is unnecessary to postulate higher-order reaction schemes for the underlying charge redistribution.

Animals↗
PubMed1983

Charge movement in a fast twitch skeletal muscle from rat.

Voltage-dependent charge movement in the rat omohyoid muscle was investigated using the three microelectrode voltage clamp technique. The charge that moved during a depolarization from the holding potential (-90 mV) to the test potential, V, increased with increasing V, saturating around 0 mV. The charge vs. voltage relationship was well fitted by Q = Q(max)/{1 + exp[-(V - V)/k]}, with Q(max) = 28.5 nC/muF, V = -34.2 mV, and k = 8.7 mV. Repolarization of the fiber from the test potential back to the holding potential caused an equal but opposite amount of charge to move. The kinetics of ON charge movement could be well described by a model developed for frog muscle by Horowicz and Schneider (1981b), which suggests that rat and frog charge movements are similar. This model failed to describe the kinetics of OFF charge movement for steps in potential from 0 mV to test potentials of -10 to -90 mV. OFF-charge movement rose to a peak more slowly and decayed more slowly than predicted by the theory.

Animals↗
PubMed1983

A quantitative study of potassium channel kinetics in rat skeletal muscle from 1 to 37 degrees C.

Potassium currents were measured using the three-microelectrode voltage-clamp technique in rat omohyoid muscle at temperatures from 1 to 37 degrees C. The currents were fitted according to the Hodgkin-Huxley equations as modified for K currents in frog skeletal muscle (Adrian et al., 1970a). The equations provided an approximate description of the time course of activation, the voltage dependence of the time constant of activation (tau n), and the voltage dependence of gK infinity. At higher temperatures the relationship between gK infinity and voltage was shifted in the hyperpolarizing direction. The effect of temperature on tau n was much greater in the cold than in the warm: tau n had a Q10 of nearly 6 at temperatures below 10 degrees C, but a Q10 of only approximately 2 over the range of 30-38 degrees C. The decreasing dependence of tau n on temperature was gradual and the Arrhenius plot of tau n revealed no obvious break-points. In addition to its quantitative effect on activation kinetics, temperature also had a qualitative effect. Near physiological temperatures (above approximately 25 degrees C), the current was well described by n4 kinetics. At intermediate temperatures (approximately 15-25 degrees C), the current was well described by n4 kinetics, but only if the n4 curve was translated rightward along the time axis (i.e., the current had a greater delay than could be accounted for by simple n4 kinetics). At low temperatures (below approximately 15 degrees C), n4 kinetics provided only an approximate fit whether or not the theoretical curve was translated along the time axis. In particular, currents in the cold displayed an initial rapid phase of activation followed by a much slower one. Thus, low temperatures appear to reveal steps in the gating process which are kinetically "hidden" at higher temperatures. Taken together, the effects of temperature on potassium currents in rat skeletal muscle demonstrate that the behavior of potassium channels at physiological temperatures cannot be extrapolated, either quantitatively or qualitatively, from experiments carried out in the cold.

Animals↗
PubMed1983

Slow components of potassium tail currents in rat skeletal muscle.

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.

Animals↗
PubMed1983

Calcium currents in a fast-twitch skeletal muscle of the rat.

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.

Animals↗
PubMed1979

Hormonally regulated phosphoprotein of turkey erythrocytes: localization to plasma membrane.

The catecholamine-stimulated cotransport of sodium and potassium ions across the plasma membrane of the turkey erythrocyte was previously found to be associated with increased 32P incorporation into a high molecular weight protein. To determine the subcellular localization of this phosphorylated protein, which we have termed goblin, a new method has been developed for isolation of pure plasma membranes from turkey erythrocytes. With this method, it has been demonstrated that goblin is located in the plasma membrane. Goblin is not extracted by solutions of low or high ionic strength but is partially extracted by nonionic detergents, indicating that it is not a component of turkey erythrocyte spectrin and suggesting that it may be an intrinsic protein of the plasma membrane. The data are compatible with a possible role for goblin in the hormonal control of ion movements across the plasma membrane.

Animals↗