Ion-concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscular junction.

The effects of deuterium oxide (D2O) and temperature on the properties of endplate channels were studied in voltage-clamped muscle fibers from the frog Rana pipiens. Studies were performed at temperatures of 8, 12, 16, and 20 degrees C. The single channel conductance (gamma) and mean channel lifetime (tau) were calculated from fluctuation analysis of the acetylcholine-induced end-plate currents. The reversal potential was determined by interpolation of the acetylcholine-induced current-voltage relation. The mean reversal potential was slightly more negative in D2O Ringer's (-7.9 +/- 0.1 mV [+/- SEM]) compared with H2O Ringer's (-5.2 +/- 0.6 mV, P less than 0.01). The single channel conductance was decreased in D2O. This decrease was greater than could be accounted for by the increased viscosity of D2O solutions, and the amount of the decrease was greater at higher temperatures. For example, gamma was 38.4 +/- 1.3 pS (+/- SEM) in H2O Ringer's and 25.7 +/- 1.0 pS in D2O Ringer's for a holding potential of -70 mV at 12 degrees C. The mean channel lifetime was significantly shorter in D2O, and the effect was greater at lower temperatures. There was not a strong effect of solvent on the temperature dependence of gamma. On the other hand, the temperature dependence of the reciprocal mean channel lifetime, alpha (where alpha = 1/tau), was strongly dependent upon the solvent. The single channel conductances showed no demonstrable voltage dependence over the range of -90 to -50 mV in both solvents. The reciprocal mean channel lifetime showed a voltage dependence, which could be described by the relation alpha = B exp(AV). The slope A was not strongly affected by either temperature or the solvent. On the other hand, the intercept B was a strong function of temperature and was weakly dependent upon the solvent, with most values greater in D2O. The D2O effects on alpha were what would be expected if they were due to the properties of D2O as a solvent (solvent isotope effects), while the D2O effects on gamma must also include the exchange of D for H in the vicinity of the selectivity filter (primary and/or secondary kinetic isotope effects).

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Veratridine modification of the purified sodium channel alpha-polypeptide from eel electroplax.

The Journal of General Physiology ◽

10.1085/jgp.94.5.813 ◽

1989 ◽

Vol 94 (5) ◽

pp. 813-831 ◽

Cited By ~ 7

Author(s):

D S Duch ◽

E Recio-Pinto ◽

C Frenkel ◽

S R Levinson ◽

B W Urban

Keyword(s):

Sodium Channel ◽

Lipid Bilayers ◽

Sodium Channels ◽

Single Channel ◽

Reversal Potential ◽

Channel Structure ◽

Dependent Manner ◽

Channel Conductance ◽

Single Channel Conductance ◽

Protein Subunit

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. Journal of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.

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The 5-hydroxytryptamine type 3 (5-HT3) receptor reveals a novel determinant of single-channel conductance

Biochemical Society Transactions ◽

10.1042/bst0320547 ◽

2004 ◽

Vol 32 (3) ◽

pp. 547-552 ◽

Cited By ~ 20

Author(s):

J.A. Peters ◽

S.P. Kelley ◽

J.I. Dunlop ◽

E.F. Kirkness ◽

T.G. Hales ◽

...

Keyword(s):

Ion Channels ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Single Channel ◽

Channel Conductance ◽

Single Channel Conductance ◽

Nicotinic Acetylcholine ◽

Ultrastructural Studies ◽

Arginine Residues ◽

Type 3

5-HT3 (5-hydroxytryptamine type 3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Two 5-HT3 receptor subunits, 5-HT3A and 5-HT3B, have been characterized in detail, although additional putative 5-HT3 subunit genes (HTR3C, HTR3D and HTR3E) have recently been reported. 5-HT3 receptors function as homopentameric assemblies of the 5-HT3 subunit, or heteropentamers of 5-HT3A and 5-HT3B subunits of unknown stoichiometry. The single-channel conductances of human recombinant homomeric and heteromeric 5-HT3 receptors are markedly different, being <1 and approx. 16 pS respectively. Paradoxically, from the results of studies performed on the closely related nicotinic acetylcholine receptor, the channel-lining M2 domain of the 5-HT3A subunit is predicted to enhance cation conduction, whereas that of the 5-HT3B subunit would not. The present study describes a novel determinant of single-channel conductance, outwith the M2 domain, which accounts for this anomaly. Utilizing a panel of chimaeric 5-HT3A and 5-HT3B subunits, a profound determinant of single-channel conductance was traced to a putative amphipathic helix (the ‘HA stretch’) within the large cytoplasmic loop of the receptor. Replacement of three arginine residues (R432, R436 and R440) unique to the HA stretch of the 5-HT3A subunit with the aligned residues (Q395, D399 and A403) of the 5-HT3B subunit increased the single-channel conductance 28-fold. Significantly, from ultrastructural studies of the Torpedo nicotinic acetylcholine receptor, the key residues may be components of narrow openings within the inner vestibule of the channel, located in the cytoplasm, which contribute to the permeation pathway. Our findings indicate an important and hitherto unappreciated function for the HA stretch in the Cys-loop family of transmitter-gated ion channels.

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Ion channels of biological membranes: prediction of single channel conductance

Theoretical Chemistry Accounts ◽

10.1007/s002140050414 ◽

1999 ◽

Vol 101 (1-3) ◽

pp. 97-102 ◽

Cited By ~ 7

Author(s):

Kishani M. Ranatunga ◽

Charlotte Adcock ◽

Ian D. Kerr ◽

Graham R. Smith ◽

Mark S. P. Sansom

Keyword(s):

Ion Channels ◽

Single Channel ◽

Biological Membranes ◽

Channel Conductance ◽

Single Channel Conductance

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Synaptic transmission : ion concentration changes in the synaptic cleft

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1979.0095 ◽

1979 ◽

Vol 206 (1162) ◽

pp. 115-131 ◽

Cited By ~ 10

Keyword(s):

Neuromuscular Junction ◽

Time Course ◽

Reversal Potential ◽

Synaptic Cleft ◽

Time Dependent ◽

Ion Concentration ◽

Frog Neuromuscular Junction ◽

Synaptic Current ◽

Sodium Depletion ◽

Concentration Changes

Currents flowing through the postsynaptic membrane of an active synapse will tend to change the concentrations of ions in the synaptic cleft. Published experimental data are used to predict ( a ) the sodium and potassium concentration changes in the cleft at the frog neuromuscular junction, and ( b ) the sodium depletion in the cleft under a Ia synaptic bouton on a cat motoneuron. Significant concentration changes are predicted at both synapses. These changes will contribute to the time dependence of the observed current and will cause the reversal potential of the current to be time dependent. At the frog neuromuscular junction, the time course of the endplate current has been shown previously to depend on the magnitude of the current flowing (at a given potential). We attribute this to changes of the cleft ion concentration. The time dependent changes of the endplate current reversal potential that we predict for the neuromuscular junction are probably too small to be detected. This is because the effects of sodium depletion and potassium accumulation on the reversal potential almost cancel. We predict that near the reversal potential small currents of complex time course will remain, i. e. no true reversal potential exists. Such currents have previously been seen experimentally. At the cat Ia synapse, the synaptic current is predicted to deplete a significant fraction of the available extracellular sodium ions. Consequently, the magnitude of the synaptic current should be relatively independent of the number of postsynaptic channels activated, and of the membrane potential, as has previously been found experimentally.

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Lack of negative slope in I-V plots for BK channels at positive potentials in the absence of intracellular blockers

The Journal of General Physiology ◽

10.1085/jgp.201210955 ◽

2013 ◽

Vol 141 (4) ◽

pp. 493-497 ◽

Cited By ~ 5

Author(s):

Yanyan Geng ◽

Xiaoyu Wang ◽

Karl L. Magleby

Keyword(s):

Single Channel ◽

Bk Channels ◽

Negative Slope ◽

Channel Conductance ◽

Single Channel Conductance ◽

Linear Current ◽

Joint Application ◽

Current Voltage ◽

Α Subunits

Large-conductance, voltage- and Ca2+-activated K+ (BK) channels display near linear current–voltage (I-V) plots for voltages between −100 and +100 mV, with an increasing sublinearity for more positive potentials. As is the case for many types of channels, BK channels are blocked at positive potentials by intracellular Ca2+ and Mg2+. This fast block progressively reduces single-channel conductance with increasing voltage, giving rise to a negative slope in the I-V plots beyond about +120 mV, depending on the concentration of the blockers. In contrast to these observations of pronounced differences in the magnitudes and shapes of I-V plots in the absence and presence of intracellular blockers, Schroeder and Hansen (2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) have reported identical I-V plots in the absence and presence of blockers for BK channels, with both plots having reduced conductance and negative slopes, as expected for blockers. Schroeder and Hansen included both Ca2+ and Mg2+ in the intracellular solution rather than a single blocker, and they also studied BK channels expressed from α plus β1 subunits, whereas most previous studies used only α subunits. Although it seems unlikely that these experimental differences would account for the differences in findings between previous studies and those of Schroeder and Hansen, we repeated the experiments using BK channels comprised of α plus β1 subunits with joint application of 2.5 mM Ca2+ plus 2.5 mM Mg2+, as Schroeder and Hansen did. In contrast to the findings of Schroeder and Hansen of identical I-V plots, we found marked differences in the single-channel I-V plots in the absence and presence of blockers. Consistent with previous studies, we found near linear I-V plots in the absence of blockers and greatly reduced currents and negative slopes in the presence of blockers. Hence, studies of conductance mechanisms for BK channels should exclude intracellular Ca2+/Mg2+, as they can reduce conductance and induce negative slopes.

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