scholarly journals Single channel studies of the phosphorylation of K+ channels in the squid giant axon. II. Nonstationary conditions.

1991 ◽  
Vol 98 (1) ◽  
pp. 19-34 ◽  
Author(s):  
E Perozo ◽  
D S Jong ◽  
F Bezanilla
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
Delayed Rectifier ◽  
Activation Process ◽  
Inactivation Curve ◽  
Caged Atp ◽  
Delayed Rectifier Current ◽  
Voltage Curve

The effects of phosphorylation on the properties of the 20-pS channel of the squid giant axon were studied using the cut-open axon technique. Phosphorylation of the channel was achieved by photoreleasing caged ATP (inside the patch pipette) in the presence of the catalytic subunit of the protein kinase A. An inverted K+ gradient (500 K+ external parallel 5 K+ internal) was used to study the activation process. Phosphorylation decreased the frequency of openings of the channel at most potentials by shifting the probability vs. voltage curve toward more positive potentials. The mean open times showed no voltage dependence and were not affected by phosphorylation. The distribution of first latencies, on the other hand, displayed a sharp voltage dependence. Phosphorylation increased the latency to the first opening at all potentials, shifting the median first latency vs. voltage curve toward more positive potentials. The slow inactivation process was studied in the presence of a physiological K+ gradient (10 K+ external parallel 310 K+ internal). Pulses to 40 mV from different holding potentials were analyzed. Phosphorylation increases the overall ensemble probability by decreasing the number of blank traces. A single channel inactivation curve was constructed by computing the relative appearance of blank traces at different holding potentials before and after photoreleasing caged ATP. As determined in dialyzed axons, the effect of phosphorylation consisted in a shift of the inactivation curve toward more positive potentials. The 20-pS channel has the same characteristics as the delayed rectifier current in activation kinetics, steady-state inactivation, and phosphorylation effects.

scholarly journals Single channel studies of the phosphorylation of K+ channels in the squid giant axon. I. Steady-state conditions.

1991 ◽  
Vol 98 (1) ◽  
pp. 1-17 ◽  
Author(s):  
E Perozo ◽  
C A Vandenberg ◽  
D S Jong ◽  
F Bezanilla
Keyword(s):  
Steady State ◽  
Single Channel ◽  
Slow Component ◽  
Giant Axon ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Delayed Rectifier Current ◽  
K Current ◽  
The Difference ◽  
Single Channel Currents

Phosphorylation of the delayed rectifier channel of squid potentiates the macroscopic K+ current and slows its activation kinetics. We have studied this phenomenon at the single channel level using the cut-open axon technique under steady-state conditions. In 10 mM external K+/310 mM internal K+ there are predominantly two types of channels present, a 20-pS and a 40-pS channel. In steady state at depolarized potentials, the 40-pS channel was most active, whereas the 20-pS channel tended to disappear due to a slow inactivation process. Two methods were developed to shift the population of channels toward a dephosphorylated state. One method consisted of predialyzing a whole axon with solutions containing no ATP, while recording the currents under axial-wire voltage clamp. A piece of axon was then removed and cut open, and single channel currents were recorded from the cut-open axon. A second method was based on the difference in diffusion coefficients for ATP and proteins such as the endogenous phosphatase. The axon was cut open in a solution that did not contain Ca2+ or Cl- in order to maintain the axoplasm structurally intact and permit endogenous phosphatase to act on the membrane while ATP diffused away, before removing the axoplasm and forming a membrane patch. When dephosphorylating conditions were used, the steady-state open probability of the 40-pS channel at 42 mV was very low (less than 0.0002), and the channel openings appeared as a series of infrequent, short-duration events. The channel activity was increased up to 150-fold by photoreleasing caged ATP inside the patch pipette in the presence of the catalytic subunit of protein kinase A. The sharp increase in open probability could be accounted for by a decrease of the slow component of the closed time distribution from 23 s to 170 ms with little change in the distribution of open times (1-2 ms) and no change in the single channel current amplitude. In voltage-jump experiments the contribution of the 40-pS channel to the delayed rectifier current was often small due to the large values of the latency to the first opening.

scholarly journals Potassium conductance of the squid giant axon. Single-channel studies.

1988 ◽  
Vol 92 (2) ◽  
pp. 179-196 ◽  
Author(s):  
I Llano ◽  
C K Webb ◽  
F Bezanilla
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Potassium Conductance ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
K Channel ◽  
Delayed Rectifier ◽  
Fluctuation Analysis ◽  
Patch Clamp Technique ◽  
Relative Contribution

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.

scholarly journals Shaker potassium channel gating. II: Transitions in the activation pathway.

1994 ◽  
Vol 103 (2) ◽  
pp. 279-319 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
J Dittman ◽  
R W Aldrich
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ionic Current ◽  
Activation Process ◽  
Charge Movement ◽  
Open Probability ◽  
Current Time ◽  
Gating Charge

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.

Cloning and characterization of a Kv1.5 delayed rectifier K+ channel from vascular and visceral smooth muscles

1994 ◽  
Vol 267 (5) ◽  
pp. C1231-C1238 ◽  
Author(s):  
K. E. Overturf ◽  
S. N. Russell ◽  
A. Carl ◽  
F. Vogalis ◽  
P. J. Hart ◽  
...  
Keyword(s):  
Single Channel ◽  
Smooth Muscles ◽  
Functional Expression ◽  
K Channel ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Transmembrane Segments ◽  
Delayed Rectifier Current ◽  
Current Voltage Curve ◽  
High Level

We have cloned and characterized the expression of a Kv1.5 K+ channel (cKv1.5) from canine colonic smooth muscle. The amino acid sequence displayed a high level of identity to other K+ channels of the Kv1.5 class in the core region between transmembrane segments S1-S6; however, identity decreased to between 74 and 82% in the NH2 and COOH terminal segments, suggesting that cKv1.5 is a distinct isoform of the Kv1.5 class. Functional expression of cKv1.5 in oocytes demonstrated a channel highly selective for K+, which activates in a voltage-dependent manner on depolarization to membrane potentials positive to -40 mV. At room temperature the channel showed fast activation (time to half of peak current, 5.5 ms) and slow inactivation that was incomplete after 20-s depolarizations. Single channel analysis of the channel expressed in oocytes displayed a linear current-voltage curve and had a slope conductance of 9.8 +/- 1.1 pS. Northern blot analysis demonstrated differential expression of cKv1.5 in smooth muscles of the gastrointestinal tract and abundant expression in several vascular smooth muscles. We propose that cKv1.5 represents a component of the delayed rectifier current in both vascular and visceral smooth muscles.

scholarly journals Uncoupling Charge Movement from Channel Opening in Voltage-gated Potassium Channels by Ruthenium Complexes

2011 ◽  
Vol 286 (18) ◽  
pp. 16414-16425 ◽  
Author(s):  
Andrés Jara-Oseguera ◽  
Itzel G. Ishida ◽  
Gisela E. Rangel-Yescas ◽  
Noel Espinosa-Jalapa ◽  
José A. Pérez-Guzmán ◽  
...  
Keyword(s):  
Electromechanical Coupling ◽  
Voltage Dependence ◽  
Voltage Sensor ◽  
Delayed Rectifier ◽  
Charge Movement ◽  
Β Cells ◽  
Delayed Rectifier Current ◽  
Mechanism Of Inhibition ◽  
Channel Opening ◽  
Sensor Activation

The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K+-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K+-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling.

Reduced voltage dependence of inactivation in the SCN5A sodium channel mutation delF1617

2005 ◽  
Vol 288 (6) ◽  
pp. H2666-H2676 ◽  
Author(s):  
Tiehua Chen ◽  
Masashi Inoue ◽  
Michael F. Sheets
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Expression System ◽  
Single Amino Acid ◽  
Wild Type ◽  
Gating Charge ◽  
Voltage Curve ◽  
Voltage Dependent ◽  
Single Channel Currents

Deletion of a phenylalanine at position 1617 (delF1617) in the extracellular linker between segments S3 and S4 in domain IV of the human heart Na+ channel (hH1a) has been tentatively associated with long QT syndrome type 3 (LQT3). In a mammalian cell expression system, we compared whole cell, gating, and single-channel currents of delF1617 with those of wild-type hH1a. The half points of the peak activation-voltage curve for the two channels were similar, as were the deactivation time constants at hyperpolarized test potentials. However, delF1617 demonstrated a significant negative shift of −7 mV in the half point of the voltage-dependent Na+ channel availability curve compared with wild type. In addition, both the time course of decay of Na+ current ( INa) and two-pulse development of inactivation of delF1617 were faster at negative test potentials, whereas they tended to be slower at positive potentials compared with wild type. Mean channel open times for delF1617 were shorter at potentials <0 mV, whereas they were longer at potentials >0 mV compared with wild type. Using anthopleurin-A, a site-3 toxin that inhibits movement of segment S4 in domain IV (S4-DIV), we found that gating charge contributed by the S4-DIV in delF1617 was reduced 37% compared with wild type. We conclude that deletion of a single amino acid in the S3-S4 linker of domain IV alters the voltage dependence of fast inactivation via a reduction in the gating charge contributed by S4-DIV and can cause either a gain or loss of INa, depending on membrane potential.

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