scholarly journals Fast and slow steps in the activation of sodium channels.

1979 ◽  
Vol 74 (6) ◽  
pp. 691-711 ◽  
Author(s):  
C M Armstrong ◽  
W F Gilly
Keyword(s):  
Early Phase ◽  
Slow Component ◽  
Na Channel ◽  
Gating Current ◽  
Similar Time ◽  
Voltage Range ◽  
Subsequent Step ◽  
Time Courses ◽  
Kinetics Of ◽  
Kinetic Features

Kinetic features of sodium conductance (gNa) and associated gating current (Ig) were studied in voltage-clamped, internally perfused squid axons. Following a step depolarization Ig ON has several kinetic components: (a) a rapid, early phase largely preceding gNa turn-on; (b) a delayed intermediate component developing as gNa increases; and (c) a slow component continuing after gNa is fully activated. With small depolarizations the early phase shows a quick rise (less than 40 mus) and smooth decay; the slow component is not detectable. During large pulses all three components are present, and the earliest shows a rising phase or initial plateau lasting approximately 80 mus. Steady-state and kinetic features of Ig are minimally influenced by control pulse currents, provided controls are restricted to a sufficiently negative voltage range. Ig OFF following a strong brief pulse also shows a rising phase. A depolarizing prepulse producing gNa inactivation and Ig immobilization eliminates the rising phase of Ig OFF. gNa, the immobilized portion of Ig ON, and the rising phase reappear with similar time-courses when tested with a second depolarizing pulse after varying periods of repolarization. 30 mM external ZnCl2 delays and slows gNa activation, prolongs the rising phase, and slows the subsequent decay of Ig ON. Zn does not affect the kinetics of gNa tails or Ig OFF as channels close, however. We present a sequential kinetic model of Na channel activation, which adequately describes the observations. The rapid early phase of IgON is generated by a series of several fast steps, while the intermediate component reflects a subsequent step. The slow component is too slow to be clearly associated with gNa activation.

Two conformational states involved in the use-dependent TTX blockade of human cardiac Na+ channel

1996 ◽  
Vol 270 (6) ◽  
pp. H2029-H2037 ◽  
Author(s):  
R. Dumaine ◽  
H. A. Hartmann
Keyword(s):  
Direct Interaction ◽  
Na Channel ◽  
Fast Inactivation ◽  
Slow Recovery ◽  
Similar Time ◽  
Activated State ◽  
High Affinity Site ◽  
Action Potential Plateau ◽  
Time Courses ◽  
High Concentrations

We used a fast inactivation-deficient mutant (QQQ) of the human heart Na+ channel alpha-subunit (hH1a) to assess the influence of the inactivation gate on tetrodotoxin (TTX) use-dependent block (UDB) and postrepolarization block (PRB). PRB had similar time courses in both channels, suggesting no direct interaction of the inactivation gate with the TTX binding site. The UDB saturated with high concentrations of TTX in hH1a but not in QQQ, revealing the modulatory action of fast inactivation on UDB. TTX did not stabilize the inactivated states of QQQ, and the extra block developing during long depolarizations suggests a higher-affinity site involved in the gating of the channel. These results cannot be solely explained by a slow recovery from the block in the inactivated states. They suggest a common use-dependent block mechanism for hH1a and QQQ involving a high-affinity site. We propose that an activated state is primarily responsible for UDB during short depolarization in the range of the action potential plateau and that fast inactivation modulates the accessibility of the toxin to this site.

scholarly journals Slowing of sodium channel opening kinetics in squid axon by extracellular zinc.

1982 ◽  
Vol 79 (6) ◽  
pp. 935-964 ◽  
Author(s):  
W F Gilly ◽  
C M Armstrong
Keyword(s):  
Rate Constants ◽  
Membrane Surface ◽  
Outward Current ◽  
Forward Rate ◽  
Na Channel ◽  
Na Channels ◽  
Gating Current ◽  
Channel Opening ◽  
The Mean ◽  
Kinetics Of

The interaction of Zn ion on Na channels was studied in squid giant axons. At a concentration of 30 mM Zn2+ slows opening kinetics of Na channels with almost no alteration of closing kinetics. The effects of Zn2+ can be expressed as a "shift" of the gating parameters along the voltage axis, i.e., the amount of additional depolarization required to overcome the Zn2+ effect. In these terms the mean shifts caused by 30 mM Zn2+ were +29.5 mV for Na channel opening (on) kinetics (t1/2 on), +2 mV for closing (off) kinetics (tau off), and +8.4 mV for the gNa-V curve. Zn2+ does not change the shape of the instantaneous I-V curve for inward current, but reduces it in amplitude by a factor of or approximately 0.67. Outward current is unaffected. Effects of Zn2+ on gating current (measured in the absence of TTX) closely parallel its actions on gNa. On gating current kinetics are shifted by +27.5 mV, off kinetics by +6 mV, and the Q-V distribution by +6.5 mV. Kinetic modeling shows that Zn2+ slows the forward rate constants in activation without affecting backward rate constants. More than one of the several steps in activation must be affected. The results are not compatible with the usual simple theory of uniform fixed surface charge. They suggest instead that Zn2+ is attracted by a negatively charged element of the gating apparatus that is present at the outer membrane surface at rest, and migrates inward on activation.

scholarly journals Activation of squid axon K+ channels. Ionic and gating current studies.

1985 ◽  
Vol 85 (4) ◽  
pp. 539-554 ◽  
Author(s):  
M M White ◽  
F Bezanilla
Keyword(s):  
Steady State ◽  
General Scheme ◽  
Channel Gating ◽  
K Channel ◽  
Na Channel ◽  
Activation Process ◽  
Gating Current ◽  
Gating Currents ◽  
Similar Time ◽  
Channel Activation

We have used data obtained from measurements of ionic and gating currents to study the process of K+ channel activation in squid giant axons. A marked improvement in the recording of K+ channel gating currents (IKg) was obtained by total replacement of Cl- in the external solution by NO-3, which eliminates approximately 50% of the Na+ channel gating current with no effect on IKg. The midpoint of the steady state charge-voltage (Qrel - V) relationship is approximately 40 mV hyperpolarized to that of the steady state activation (fo - V) curve, which is an indication that the channel has many nonconducting states. Ionic and gating currents have similar time constants for both ON and OFF pulses. This eliminates any Hodgkin-Huxley nx scheme for K+ channel activation. An interrupted pulse paradigm shows that the last step in the activation process is not rate limiting. IKg shows a nonartifactual rising phase, which indicates that the first step is either the slowest step in the activation sequence or is voltage independent. These data are consistent with the following general scheme for K+ channel activation: (formula; see text)

Kinetic properties and inactivation of the gating currents of sodium channels in squid axon

1975 ◽  
Vol 270 (908) ◽  
pp. 449-458 ◽  
Keyword(s):  
Sodium Channels ◽  
Sodium Current ◽  
Kinetic Properties ◽  
Gating Current ◽  
Gating Currents ◽  
Similar Time ◽  
Sodium Permeability ◽  
Small Component ◽  
Current Decay ◽  
Time Courses

Associated with the opening and closing of the sodium channels of nerve membrane is a small component of capacitative current, the gating current. After termination of a depolarizing step the gating current and sodium current decay with similar time courses. Both currents decay more rapidly at relatively negative membrane voltages than at positive ones. The gating current that flows during a depolarizing step is diminished by a pre-pulse that inactivates the sodium permeability. A pre-pulse has no effect after inactivation has been destroyed by internal perfusion with the proteolytic enzyme pronase. Gating charge (considered as positive charge) moves outward during a positive voltage step, with voltage dependent kinetics. The time constant of the outward gating current is a maximum at about —10 mV, and has a smaller value at voltages either more positive or negative than this value.

scholarly journals Kinetic Control of Multiple Forms of Ca2+ Spikes by Inositol Trisphosphate in Pancreatic Acinar Cells

1999 ◽  
Vol 146 (2) ◽  
pp. 405-414 ◽  
Author(s):  
Koichi Ito ◽  
Yasushi Miyashita ◽  
Haruo Kasai
Keyword(s):  
Phospholipase C ◽  
Critical Role ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Kinetic Control ◽  
Multiple Forms ◽  
Similar Time ◽  
Inositol 1,4,5 Trisphosphate ◽  
Time Courses ◽  
Kinetics Of

The mechanisms of agonist-induced Ca2+ spikes have been investigated using a caged inositol 1,4,5-trisphosphate (IP3) and a low-affinity Ca2+ indicator, BTC, in pancreatic acinar cells. Rapid photolysis of caged IP3 was able to reproduce acetylcholine (ACh)-induced three forms of Ca2+ spikes: local Ca2+ spikes and submicromolar (<1 μM) and micromolar (1–15 μM) global Ca2+ spikes (Ca2+ waves). These observations indicate that subcellular gradients of IP3 sensitivity underlie all forms of ACh-induced Ca2+ spikes, and that the amplitude and extent of Ca2+ spikes are determined by the concentration of IP3. IP3-induced local Ca2+ spikes exhibited similar time courses to those generated by ACh, supporting a role for Ca2+-induced Ca2+ release in local Ca2+ spikes. In contrast, IP3- induced global Ca2+ spikes were consistently faster than those evoked with ACh at all concentrations of IP3 and ACh, suggesting that production of IP3 via phospholipase C was slow and limited the spread of the Ca2+ spikes. Indeed, gradual photolysis of caged IP3 reproduced ACh-induced slow Ca2+ spikes. Thus, local and global Ca2+ spikes involve distinct mechanisms, and the kinetics of global Ca2+ spikes depends on that of IP3 production particularly in those cells such as acinar cells where heterogeneity in IP3 sensitivity plays critical role.

scholarly journals Pseudo-streaming potentials in Necturus gallbladder epithelium. II. The mechanism is a junctional diffusion potential.

1992 ◽  
Vol 99 (3) ◽  
pp. 317-338 ◽  
Author(s):  
L Reuss ◽  
B Simon ◽  
C U Cotton
Keyword(s):  
Time Course ◽  
Bathing Solution ◽  
Intercellular Spaces ◽  
Similar Time ◽  
Streaming Potentials ◽  
Osmotic Water ◽  
Lateral Intercellular Spaces ◽  
Transepithelial Voltage ◽  
Time Courses ◽  
Good Agreement

The mechanisms of apparent streaming potentials elicited across Necturus gallbladder epithelium by addition or removal of sucrose from the apical bathing solution were studied by assessing the time courses of: (a) the change in transepithelial voltage (Vms). (b) the change in osmolality at the cell surface (estimated with a tetrabutylammonium [TBA+]-selective microelectrode, using TBA+ as a tracer for sucrose), and (c) the change in cell impermeant solute concentration ([TMA+]i, measured with an intracellular double-barrel TMA(+)-selective microelectrode after loading the cells with TMA+ by transient permeabilization with nystatin). For both sucrose addition and removal, the time courses of Vms were the same as the time courses of the voltage signals produced by [TMA+]i, while the time courses of the voltage signals produced by [TBA+]o were much faster. These results suggest that the apparent streaming potentials are caused by changes of [NaCl] in the lateral intercellular spaces, whose time course reflects the changes in cell water volume (and osmolality) elicited by the alterations in apical solution osmolality. Changes in cell osmolality are slow relative to those of the apical solution osmolality, whereas lateral space osmolality follows cell osmolality rapidly, due to the large surface area of lateral membranes and the small volume of the spaces. Analysis of a simple mathematical model of the epithelium yields an apical membrane Lp in good agreement with previous measurements and suggests that elevations of the apical solution osmolality elicit rapid reductions in junctional ionic selectivity, also in good agreement with experimental determinations. Elevations in apical solution [NaCl] cause biphasic transepithelial voltage changes: a rapid negative Vms change of similar time course to that of a Na+/TBA+ bi-ionic potential and a slow positive Vms change of similar time course to that of the sucrose-induced apparent streaming potential. We conclude that the Vms changes elicited by addition of impermeant solute to the apical bathing solution are pseudo-streaming potentials, i.e., junctional diffusion potentials caused by salt concentration changes in the lateral intercellular spaces secondary to osmotic water flow from the cells to the apical bathing solution and from the lateral intercellular spaces to the cells. Our results do not support the notion of junctional solute-solvent coupling during transepithelial osmotic water flow.

scholarly journals Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 475-488 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Katja Kohler ◽  
Andrea Bacconi ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Reaction Rates ◽  
Transport Pathway ◽  
Extracellular Medium ◽  
Voltage Range ◽  
Substituted Cysteine Accessibility ◽  
Voltage Dependent ◽  
Opposite Behavior ◽  
Rate Limiting ◽  
Kinetics Of

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

Basis for tetrodotoxin and lidocaine effects on action potentials in dog ventricular myocytes

1988 ◽  
Vol 254 (6) ◽  
pp. H1157-H1166 ◽  
Author(s):  
J. A. Wasserstrom ◽  
J. J. Salata
Keyword(s):  
Action Potential ◽  
Action Potentials ◽  
Ventricular Myocytes ◽  
Ionic Currents ◽  
Detectable Effect ◽  
Na Channel ◽  
Na Current ◽  
Voltage Range ◽  
Purkinje Fibers ◽  
Ventricular Cells

We studied the effects of tetrodotoxin (TTX) and lidocaine on transmembrane action potentials and ionic currents in dog isolated ventricular myocytes. TTX (0.1-1 x 10(-5) M) and lidocaine (0.5-2 x 10(-5) M) decreased action potential duration, but only TTX decreased the maximum rate of depolarization (Vmax). Both TTX (1-2 x 10(-5) M) and lidocaine (2-5 x 10(-5) M) blocked a slowly inactivating toward current in the plateau voltage range. The voltage- and time-dependent characteristics of this current are virtually identical to those described in Purkinje fibers for the slowly inactivating inward Na+ current. In addition, TTX abolished the outward shift in net current at plateau potentials caused by lidocaine alone. Lidocaine had no detectable effect on the slow inward Ca2+ current and the inward K+ current rectifier, Ia. Our results indicate that 1) there is a slowly inactivating inward Na+ current in ventricular cells similar in time, voltage, and TTX sensitivity to that described in Purkinje fibers; 2) both TTX and lidocaine shorten ventricular action potentials by reducing this slowly inactivating Na+ current; 3) lidocaine has no additional actions on other ionic currents that contribute to its ability to abbreviate ventricular action potentials; and 4) although both agents shorten the action potential by the same mechanism, only TTX reduces Vmax. This last point suggests that TTX produces tonic block of Na+ current, whereas lidocaine may produce state-dependent Na+ channel block, namely, blockade of Na+ current only after Na+ channels have already been opened (inactivated-state block).

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