scholarly journals Sodium Flux Ratio in Na/K Pump-Channels Opened by Palytoxin

2007 ◽  
Vol 130 (1) ◽  
pp. 41-54 ◽  
Author(s):  
R.F. Rakowski ◽  
Pablo Artigas ◽  
Francisco Palma ◽  
Miguel Holmgren ◽  
Paul De Weer ◽  
...  
Keyword(s):  
Binding Sites ◽  
Reversal Potential ◽  
Flux Ratio ◽  
Giant Fiber ◽  
Animal Cells ◽  
Giant Axons ◽  
Single File ◽  
Sodium Flux ◽  
Na Ions ◽  
Loligo Pealei

Palytoxin binds to Na+/K+ pumps in the plasma membrane of animal cells and opens an electrodiffusive cation pathway through the pumps. We investigated properties of the palytoxin-opened channels by recording macroscopic and microscopic currents in cell bodies of neurons from the giant fiber lobe, and by simultaneously measuring net current and 22Na+ efflux in voltage-clamped, internally dialyzed giant axons of the squid Loligo pealei. The conductance of single palytoxin-bound “pump-channels” in outside-out patches was ∼7 pS in symmetrical 500 mM [Na+], comparable to findings in other cells. In these high-[Na+], K+-free solutions, with 5 mM cytoplasmic [ATP], the K0.5 for palytoxin action was ∼70 pM. The pump-channels were ∼40–50 times less permeable to N-methyl-d-glucamine (NMG+) than to Na+. The reversal potential of palytoxin-elicited current under biionic conditions, with the same concentration of a different permeant cation on each side of the membrane, was independent of the concentration of those ions over the range 55–550 mM. In giant axons, the Ussing flux ratio exponent (n') for Na+ movements through palytoxin-bound pump-channels, over a 100–400 mM range of external [Na+] and 0 to −40 mV range of membrane potentials, averaged 1.05 ± 0.02 (n = 28). These findings are consistent with occupancy of palytoxin-bound Na+/K+ pump-channels either by a single Na+ ion or by two Na+ ions as might be anticipated from other work; idiosyncratic constraints are needed if the two Na+ ions occupy a single-file pore, but not if they occupy side-by-side binding sites, as observed in related structures, and if only one of the sites is readily accessible from both sides of the membrane.

scholarly journals Sodium flux ratio in voltage-clamped squid giant axons.

1981 ◽  
Vol 77 (5) ◽  
pp. 489-502 ◽  
Author(s):  
T Begenisich ◽  
D Busath
Keyword(s):  
Sodium Channels ◽  
Binding Sites ◽  
Flux Ratio ◽  
Ion Binding ◽  
Ion Permeation ◽  
Giant Axons ◽  
Sodium Flux ◽  
Ion Binding Sites ◽  
Loligo Pealei ◽  
Ion Pore

The sodium flux ratio across the axolemma of internally perfused, voltage-clamped giant axons of Loligo pealei has been measured at various membrane potentials. The flux ratio exponent obtained from these measurements was about unity and independent of membrane voltage over the 50 mV range from about -20 to +30 mV. These results, combined with previous measurements of ion permeation through sodium channels, show that the sodium channel behaves like a multi-ion pore with two ion binding sites that are rarely simultaneously occupied by sodium.

scholarly journals Potassium flux ratio in voltage-clamped squid giant axons.

1980 ◽  
Vol 76 (1) ◽  
pp. 83-98 ◽  
Author(s):  
T Begenisich ◽  
P De Weer
Keyword(s):  
Simultaneous Measurement ◽  
Potassium Conductance ◽  
Flux Ratio ◽  
Giant Axon ◽  
Equilibrium Potential ◽  
Giant Axons ◽  
Average Value ◽  
Potassium Flux ◽  
K Current ◽  
Loligo Pealei

The potassium flux ratio across the axolemma of internally perfused, voltage-clamped giant axons of Loligo pealei has been evaluated at various membrane potentials and internal potassium concentrations ([K]i). Four different methods were used: (a) independent measurement of one-way influx and efflux of 42K; (b) simultaneous measurement of net K current (IK) and 42K influx; (c) simultaneous measurement of IK and 42K efflux; and (d) measurement of potassium conductance and 42K influx at the potassium equilibrium potential. The reliability of each of these methods is discussed. The average value of the exponent n' in the Hodgkin-Keynes equation ranged from 1.5 at -4mV and 200 mM [K]i to 3.3 at -38 mV and 350 mM [K]i and appeared to be a function of membrane potential and possibly of [K]i. It is concluded that the potassium channel of squid giant axon is a multi-ion, single-file pore with three or more sites.

scholarly journals Molecular identification of SqKv1A. A candidate for the delayed rectifier K channel in squid giant axon.

1996 ◽  
Vol 108 (3) ◽  
pp. 207-219 ◽  
Author(s):  
J J Rosenthal ◽  
R G Vickery ◽  
W F Gilly
Keyword(s):  
Time Course ◽  
Xenopus Oocytes ◽  
Giant Axon ◽  
K Channel ◽  
Delayed Rectifier ◽  
Giant Fiber ◽  
Western Blots ◽  
Internal Solution ◽  
Giant Axons ◽  
K Currents

We have cloned the cDNA for a squid Kvl potassium channel (SqKv1A). SqKv1A mRNA is selectively expressed in giant fiber lobe (GFL) neurons, the somata of the giant axons. Western blots detect two forms of SqKv1A in both GFL neuron and giant axon samples. Functional properties of SqKv1A currents expressed in Xenopus oocytes are very similar to macroscopic currents in GFL neurons and giant axons. Macroscopic K currents in GFL neuron cell bodies, giant axons, and in Xenopus oocytes expressing SqKv1A, activate rapidly and inactivate incompletely over a time course of several hundred ms. Oocytes injected with SqKv1A cRNA express channels of two conductance classes, estimated to be 13 and 20 pS in an internal solution containing 470 mM K. SqKv1A is thus a good candidate for the "20 pS" K channel that accounts for the majority of rapidly activating K conductance in both GFL neuron cell bodies and the giant axon.

Tracer Na fluxes in Necturus proximal tubule

1977 ◽  
Vol 232 (5) ◽  
pp. F461-F470 ◽  
Author(s):  
K. R. Spring ◽  
G. Giebisch
Keyword(s):  
Flux Ratio ◽  
Cell Membrane Permeability ◽  
Plasma Flux ◽  
Capillary Perfusion ◽  
Sodium Permeability ◽  
Shunt Pathway ◽  
New Methods ◽  
Sodium Flux ◽  
Nonsteady State ◽  
Necturus Proximal Tubule

Steady-state bidirectional sodium fluxes were measured across Necturus proximal tubules. New methods for capillary perfusion and collection of venous effluent enabled flux determination to be made from the appearance of luminal tracer in the capillaries. Fluxes and permeability were measured in the absence of net fluid reabsorption. The sodium permeability measured in the plasma-to-lumen direction was 3 X 10(-6) cm/s. The flux ratio (lumen-to-plasma/plasma-to-lumen) was about twice the passive value calculated from the measured concentrations and potentials. Estimates for the permeability and flux across the shunt pathway were obtained from nonsteady-state flux determinations. The shunt pathway appeared to be the most significant route for passive sodium movement from plasma-to-lumen. Nonsteady-state tracer measurements also enabled an estimate to be made of the lumenal cell membrane permeability and unidirectional sodium flux. Two-thirds of the lumen-to-plasma flux was calculated to traverse the cellular path and the remainder through the shunt. Approximately one-third of the intracellular sodium was found to exchange rapidly with tracer.

scholarly journals Binding of nitrobenzylthioinosine to high-affinity sites on the nucleoside-transport mechanism of HeLa cells

1981 ◽  
Vol 200 (2) ◽  
pp. 295-305 ◽  
Author(s):  
E Dahlig-Harley ◽  
Y Eilam ◽  
A R P Paterson ◽  
C E Cass
Keyword(s):  
Hela Cells ◽  
Binding Sites ◽  
Monolayer Culture ◽  
Nucleoside Transport ◽  
Thiol Groups ◽  
Transport Activity ◽  
Animal Cells ◽  
High Affinity ◽  
Effect Curve ◽  
Km Value

Nitrobenzylthioinosine (NBMPR) binds reversibly, but with high affinity (Kd 0.1--1.2 nM), to inhibitory sites on nucleoside-transport elements of the plasma membrane in a variety of animal cells. The present study explored relationships in HeLa cells between NBMPR binding and inhibition of uridine transport. The Km value for inward transport of uridine by HeLa cells in both suspension and monolayer culture was about 0.1 mM. The affinity of the transport-inhibitory sites for uridine (Kd 1.7 mM), inosine (Kd 0.4 mM) and other nucleoside permeants was low relative to that for NBMPR. The pyrimidine homologue of NBMPR, nitrobenzylthiouridine, also exhibited low affinity for the NBMPR-binding sites. Pretreatment of HeLa cells with p-chloromercuribenzene sulphonate (p-CMBS) or N-ethylmaleimide (NEM) decreased binding of NBMPR to its high-affinity sites and inhibited uridine transport, indicating the presence of thiol groups essential to both processes. NEM, a more penetrable reagent than p-CMBS, inhibited binding and transport at much lower concentrations than the latter compound. Pretreatment of cells with concentrations of p-CMBS that alone had no effect on either NBMPR binding or uridine transport increased the sensitivity of transport to NBMPR inhibition and changed the shape of the NBMPR concentration-effect curve, suggesting synergistic inhibiton of uridine-transport activity by these two agents.

Effects of extracellular sodium concentration on null potential, conductance and open time of endplate channels

1982 ◽  
Vol 216 (1203) ◽  
pp. 225-251 ◽  
Keyword(s):  
Binding Sites ◽  
Single Channel ◽  
Sodium Concentration ◽  
Potassium Ions ◽  
Channel Conductance ◽  
Channel Characteristics ◽  
Open Time ◽  
Na Ions ◽  
Channel Open Time ◽  
Extracellular Sodium

(i) Effects of extracellular sodium concentration, [Na] o , on endplate channel characteristics were investigated in voltage-clamped, glycerol- treated toad sartorius fibres. (ii) The relation between [Na] o (and [K] o ) and acetylcholine null potential could be reasonably well fitted by the Goldman-Hodgkin-Katz type of equation, except when [Na] o was higher than normal. Anions had no significant effect on the null potential. (iii) Endplate channel open time (ז), whether measured from miniature endplate currents or from current fluctuations induced by iontophoresis of acetylcholine, varied inversely with [Na] o . The relation between ז -1 (=α) and [Na] o could be fitted by α = αmax [Na] o / ( K m +|[Na] o ) with a K m of 92 mM. (iv) Endplate conductance, measured at the peak of endplate currents or at the peak of spontaneous miniature endplate currents, increased nonlinearly with [Na] o . (v) Single channel conductance, γ, also increased nonlinearly with [Na] o . Experimental observations at -90 mV could be fitted by the relation γ = γ max [Na] o / ( K m + [Na] o ), giving values for γ max and K m of 47 pS and 146 mM respectively. Correcting channel conductance for the contribution from potassium ions gave values of γmax and K m of 78 pS and 423 mM respectively. (vi) The results are consistent with the hypothesis that binding sites for Na ions can modulate both channel lifetime and conductance and that these sites become saturated at higher sodium concentrations.

Inhibition of mechanosensory interneurons in the crayfish. I. Presynaptic inhibition from giant fibers

1980 ◽  
Vol 43 (6) ◽  
pp. 1495-1509 ◽  
Author(s):  
D. Kennedy ◽  
J. McVittie ◽  
R. Calabrese ◽  
R. A. Fricke ◽  
W. Craelius ◽  
...  
Keyword(s):  
Reversal Potential ◽  
Repetitive Firing ◽  
Primary Afferent Depolarization ◽  
Gamma Aminobutyric Acid ◽  
Giant Axons ◽  
Pharmacological Studies ◽  
Sucrose Gap ◽  
Conductance Increase ◽  
Tail Flip ◽  
Stimulation Of

1. Sucrose-gap and intracellular recordings were used to study the primary afferent depolarization (PAD) produced in mechanosensory afferents by impulses in lateral and medial giant axons, which are the command cells for the tail flip escape response in the crayfish. 2. The lateral and medial giant axons produce PAD through a polysynaptic interneuronal pathway. The response has a relatively long intraganglionic latency (7--11 ms), and command-evoked PAD can be recorded in ganglia from which the giant axons have been experimentally disconnected. 3. The final neurons of the pathway that delivers inhibition are few in number and extensive in distribution; most appear to be common to lateral and medial giant pathways. 4. At least some of the inhibitory interneurons have axons in the interganglionic connectives and probably produce both presynaptic and postsynaptic inhibition. 5. Stimulation of the lateral, but not the medial, giant axons causes a small, short-latency deplorization that is stable at high repetition rates. This small potential can be accounted for by transmission across known electrical synapses between mechanosensory afferents and the lateral giants in each abdominal ganglion. 6. Repetitive stimulation of the lateral giant axons causes substantial augmentation of PAD, apparently through recruitment of additional interneurons. PAD evoked by a single medial giant (MG) stimulus is generally much larger than that elicited by a single lateral giant (LG) spike. However, MG-PAD summates little and so the maximum PAD deltaV reached during repetitive firing is equivalent for the two types of giant axons. 7. Iontophoresis of gamma-aminobutyric acid (GABA) into the ganglionic neuropil depolarizes the primary afferents and blocks activity in neurons that have axons in the interganglionic connective. 8. The extrapolated PAD reversal potential and pharmacological studies suggest that a GABA-mediated chloride conductance increase is involved in the production of PAD.

scholarly journals The prokaryotic Na+/Ca2+ exchanger NCX_Mj transports Na+ and Ca2+ in a 3:1 stoichiometry

2017 ◽  
Vol 150 (1) ◽  
pp. 51-65 ◽  
Author(s):  
Irina Shlosman ◽  
Fabrizio Marinelli ◽  
José D. Faraldo-Gómez ◽  
Joseph A. Mindell
Keyword(s):  
Binding Sites ◽  
Structural Model ◽  
Reversal Potential ◽  
Ion Binding ◽  
Experimental Conditions ◽  
Temporal Regulation ◽  
Cellular Processes ◽  
Sodium Calcium ◽  
Functional Features ◽  
Ion Binding Sites

Intracellular Ca2+ signals control a wide array of cellular processes. These signals require spatial and temporal regulation of the intracellular Ca2+ concentration, which is achieved in part by a class of ubiquitous membrane proteins known as sodium–calcium exchangers (NCXs). NCXs are secondary-active antiporters that power the translocation of Ca2+ across the cell membrane by coupling it to the flux of Na+ in the opposite direction, down an electrochemical gradient. Na+ and Ca2+ are translocated in separate steps of the antiport cycle, each of which is thought to entail a mechanism whereby ion-binding sites within the protein become alternately exposed to either side of the membrane. The prokaryotic exchanger NCX_Mj, the only member of this family with known structure, has been proposed to be a good functional and structural model of mammalian NCXs; yet our understanding of the functional properties of this protein remains incomplete. Here, we study purified NCX_Mj reconstituted into liposomes under well-controlled experimental conditions and demonstrate that this homologue indeed shares key functional features of the NCX family. Transport assays and reversal-potential measurements enable us to delineate the essential characteristics of this antiporter and establish that its ion-exchange stoichiometry is 3Na+:1Ca2+. Together with previous studies, this work confirms that NCX_Mj is a valid model system to investigate the mechanism of ion recognition and membrane transport in sodium–calcium exchangers.

scholarly journals A scheme to account for the effects of Rb+ and K+ on inward rectifier K channels of bovine artery endothelial cells.

1994 ◽  
Vol 103 (4) ◽  
pp. 549-581 ◽  
Author(s):  
P S Pennefather ◽  
T E DeCoursey
Keyword(s):  
Binding Sites ◽  
Voltage Dependence ◽  
Reversal Potential ◽  
Companion Paper ◽  
K Channel ◽  
Gating Model ◽  
Allosteric Sites ◽  
Voltage Dependent ◽  
Cooperative Process ◽  
Activator Ions

An electrochemical gating model is presented to account for the effects described in the companion paper by M. R. Silver, M. S. Shapiro, and T. E. DeCoursey (1994. Journal of General Physiology, 103:519-548) of Rb+ and Rb+/K+ mixtures on the kinetics and voltage dependence of an inwardly rectifying (IR) K+ channel. The model proposes that both Rb+ and K+ act as allosteric modulators of an intrinsically voltage dependent isomerization between open and closed states. Occupancy of binding sites on the outside of the channel promotes channel opening and stabilizes the open state. Rb+ binds to separate sites within the pore and plugs IR channels. Occupancy of the pore by Rb+ can modify the rates of isomerization and the affinity of the allosteric sites for activator ions. The model also incorporates the proposed triple-barreled nature of the IR channel (Matsuda, H., 1988. Journal of Physiology. 397:237-258.) by proposing that plugging of the channel is a cooperative process involving a single site in each of the three bores, 80% of the way through the membrane field. Interaction between bores during plugging and permeation is consistent with correlated flux models of the properties of the IR channel. Parallel bores multiply the number allosteric sites associated with the macromolecular channel and allow for steep voltage dependence without compromising the parallel shift of the half-activation potential with reversal potential. Our model proposes at least six and possibly 12 such allosteric binding sites for activator ions. We derive algebraic relations that permit derivation of parameters that define simple versions of our model from the data of Silver et al. (1994). Numerical simulations based on those parameters closely reproduce that data. The model reproduces the RS+ induced slowing of IR kinetics and the negative shift of the relation between the half-activation voltage (V1/2) and reversal potential when channel plugging is associated with (a) a slowing of the isomerization rates; (b) an increase in the affinity of allosteric sites on closed channels that promote opening; and (c) a decrease in the affinity of sites on open channels that slow closing. Rb+ also slows closing at positive potentials where open channel blockade is unlikely. Allowing Rb+ to be 1.5 times more potent than K+ as an activator in the model can account for this effect and improves the match between the predicted and observed relation between the Rb+ to K+ mole fraction and the opening rate at V1/2.(ABSTRACT TRUNCATED AT 400 WORDS)

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