scholarly journals Alternative Splicing in the Voltage-Sensing Region of N-Type CaV2.2 Channels Modulates Channel Kinetics

2004 ◽  
Vol 92 (5) ◽  
pp. 2820-2830 ◽  
Author(s):  
Yingxin Lin ◽  
Stefan I. McDonough ◽  
Diane Lipscombe
Keyword(s):  
Alternative Splicing ◽  
Cell Lines ◽  
Time Course ◽  
Voltage Dependence ◽  
Sympathetic Neurons ◽  
Charge Movement ◽  
Gating Currents ◽  
Splice Isoforms ◽  
Tsa201 Cell ◽  
Kinetics Of

The CaV2.2 gene encodes the functional core of the N-type calcium channel. This gene has the potential to generate thousands of CaV2.2 splice isoforms with different properties. However, the functional significance of most sites of alternative splicing is not established. The IVS3-IVS4 region contains an alternative splice site that is conserved evolutionarily among CaVα1 genes from Drosophila to human. In CaV2.2, inclusion of exon 31a in the IVS3-IVS4 region is restricted to the peripheral nervous system, and its inclusion slows the speed of channel activation. To investigate the effects of exon 31a in more detail, we generated four tsA201 cell lines stably expressing CaV2.2 splice isoforms. Coexpression of auxiliary CaVβ and CaVα2δ subunits was required to reconstitute currents with the kinetics of N-type channels from neurons. Channels including exon 31a activated and deactivated more slowly at all voltages. Current densities were high enough in the stable cell lines co-expressing CaVα2δ to resolve gating currents. The steady-state voltage dependence of charge movement was not consistently different between splice isoforms, but on gating currents from the exon 31a-containing CaV2.2 isoform decayed with a slower time course, corresponding to slower movement of the charge sensor. Exon 31a-containing CaV2.2 is restricted to peripheral ganglia; and the slower gating kinetics of CaV2.2 splice isoforms containing exon 31a correlated reasonably well with the properties of native N-type currents in sympathetic neurons. Our results suggest that alternative splicing in the S3-S4 linker influences the kinetics but not the voltage dependence of N-type channel gating.

scholarly journals Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

1997 ◽  
Vol 110 (5) ◽  
pp. 579-589 ◽  
Author(s):  
Riccardo Olcese ◽  
Ramón Latorre ◽  
Ligia Toro ◽  
Francisco Bezanilla ◽  
Enrico Stefani
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Ionic Current ◽  
K Channel ◽  
Charge Movement ◽  
Slow Inactivation ◽  
Gating Currents ◽  
Similar Time ◽  
K Channels ◽  
Recovery From Inactivation

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

Gating currents in the node of Ranvier: voltage and time dependence

1975 ◽  
Vol 270 (908) ◽  
pp. 483-492 ◽  
Keyword(s):  
Membrane Potential ◽  
Time Constant ◽  
Time Course ◽  
Voltage Dependence ◽  
Sudden Change ◽  
Node Of Ranvier ◽  
Ionic Currents ◽  
Gating Currents ◽  
Myelinated Nerve ◽  
Sodium Conductance

Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmann’s law (midpoint potential —33.7 mV; saturation value 17200 charges/(μm 2 ). The time course of the asymmetry current and the voltage dependence of its time constant are consistent with the notion that due to a sudden change in membrane potential the charges undergo a first order transition between two configurations. Size and voltage dependence of the time constant are similar to those of the activation of the sodium conductance assuming m 2 h kinetics, The results suggest the presence of ten times more sodium channels (5000/μm2) in the node of Ranvier than in the squid giant axon with similar sodium conductance per channel (2-3 pS),

scholarly journals A time- and voltage-dependent K+ current in single cardiac cells from bullfrog atrium.

1986 ◽  
Vol 88 (6) ◽  
pp. 777-798 ◽  
Author(s):  
J R Hume ◽  
W Giles ◽  
K Robinson ◽  
E F Shibata ◽  
R D Nathan ◽  
...  
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Outward Current ◽  
Cardiac Cells ◽  
Delayed Rectifier ◽  
Mechanical Dispersion ◽  
Atrial Cells ◽  
Voltage Dependent ◽  
K Current ◽  
Kinetics Of

Individual myocytes were isolated from bullfrog atrium by enzymatic and mechanical dispersion, and a one-microelectrode voltage clamp was used to record the slow outward K+ currents. In normal [K+]o (2.5 mM), the slow outward current tails reverse between -95 and -100 mV. This finding, and the observed 51-mV shift of Erev/10-fold change in [K+]o, strongly suggest that the "delayed rectifier" in bullfrog atrial cells is a K+ current. This current, IK, plays an important role in initiating repolarization, and it is distinct from the quasi-instantaneous, inwardly rectifying background current, IK. In atrial cells, IK does not exhibit inactivation, and very long depolarizing clamp steps (20 s) can be applied without producing extracellular K+ accumulation. The possibility of [K+]o accumulation contributing to these slow outward current changes was assessed by (a) comparing reversal potentials measured after short (2 s) and very long (15 s) activating prepulses, and (b) studying the kinetics of IK at various holding potentials and after systematically altering [K+]o. In the absence of [K+]o accumulation, the steady state activation curve (n infinity) and fully activated current-voltage (I-V) relation can be obtained directly. The threshold of the n infinity curve is near -50 mV, and it approaches a maximum at +20 mV; the half-activation point is approximately -16 mV. The fully activated I-V curve of IK is approximately linear in the range -40 to +30 mV. Semilog plots of the current tails show that each tail is a single-exponential function, which suggests that only one Hodgkin-Huxley conductance underlies this slow outward current. Quantitative analysis of the time course of onset of IK and of the corresponding envelope of tails demonstrate that the activation variable, n, must be raised to the second power to fit the sigmoid onset accurately. The voltage dependence of the kinetics of IK was studied by recording and curve-fitting activating and deactivating (tail) currents. The resulting 1/tau n curve is U-shaped and somewhat asymmetric; IK exhibits strong voltage dependence in the diastolic range of potentials. Changes in the [Ca2+]o in the superfusing Ringer's, and/or addition of La3+ to block the transmembrane Ca2+ current, show that the time course and magnitude of IK are not significantly modulated by transmembrane Ca2+ movements, i.e., by ICa. These experimentally measured voltage- and time-dependent descriptors of IK strongly suggest an important functional role for IK in atrial tissue: it initiates repolarization and can be an important determinant of rate-induced changes in action potential duration.

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.

scholarly journals Sodium channel gating currents in frog skeletal muscle.

1983 ◽  
Vol 82 (5) ◽  
pp. 679-701 ◽  
Author(s):  
D T Campbell
Keyword(s):  
Skeletal Muscle ◽  
Sodium Channel ◽  
Time Course ◽  
Channel Gating ◽  
Total Charge ◽  
Charge Movement ◽  
Frog Skeletal Muscle ◽  
Gating Currents ◽  
Gating Mechanism ◽  
Charge Movements

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.

Activation Kinetics of the Delayed Rectifier Potassium Current of Bullfrog Sympathetic Neurons

1998 ◽  
Vol 79 (5) ◽  
pp. 2345-2357 ◽  
Author(s):  
Kathryn G. Klemic ◽  
Dominique M. Durand ◽  
Stephen W. Jones
Keyword(s):  
Potassium Current ◽  
Time Course ◽  
Sympathetic Neurons ◽  
Delayed Rectifier ◽  
Activation Kinetics ◽  
Whole Cell ◽  
Delayed Rectifier Potassium Current ◽  
Kinetics Of ◽  
Cell Data ◽  
Allosteric Model

Klemic, Kathryn G., Dominique M. Durand, and Stephen W. Jones. Activation kinetics of the delayed rectifier potassium current of bullfrog sympathetic neurons. J. Neurophysiol. 79: 2345–2357, 1998. We examined the activation kinetics of the delayed rectifier K+ current of bullfrog sympathetic neurons, primarily using whole cell recording. On depolarization, currents activated with a sigmoid delay but did not show a Cole-Moore shift. The time course of activation differed systematically from an exponential raised to a power. At most voltages, a power of 2 gave the best overall fit but a power of 3 better described the initial delay. After the delay, the time course could be fitted by a single exponential. Time constants were 15–20 ms at 0 mV and decreased to a limiting τ = 7 ms at +50 to +100 mV. Tail currents were well fitted by single exponential functions and accelerated with hyperpolarization, from τ = 15–20 ms at 0 mV to τ = 2 ms at −110 mV ( e-fold for 40 mV). Eleven kinetic models were evaluated for their ability to describe the activation kinetics of the delayed rectifier. Hodgkin-Huxley–like models did not fit the data well. A linear model where voltage sensor movement is followed by a distinct channel opening step, allosteric models based on the Monod-Wyman–Changeux model, and an unconstrained C-C-C-O model could describe whole cell data from −100 to +40 mV. After including whole cell data at +60 and +80 mV, and a maximal p open of 0.8 from noise analysis of cell-attached patches, an allosteric model fit the data best, as the other models had difficulty describing qualitative features of the data. However, some more complex schemes (with additional free parameters) cannot be excluded. We propose the allosteric model as an empirical description of macroscopic ionic currents, and as a model worth considering in future studies on the molecular mechanism of potassium channel gating.

scholarly journals Inactivation of the sodium channel. II. Gating current experiments.

1977 ◽  
Vol 70 (5) ◽  
pp. 567-590 ◽  
Author(s):  
C M Armstrong ◽  
F Bezanilla
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Slow Component ◽  
Base Line ◽  
Charge Movement ◽  
Gating Current ◽  
Small Current ◽  
Gating Charge ◽  
Recovery From Inactivation ◽  
Activation Gate

Gating current (Ig) has been studied in relation to inactivation of Na channels. No component of Ig has the time course of inactivation; apparently little or no charge movement is associated with this step. Inactivation nonetheless affects Ig by immobilizing about two-thirds of gating charge. Immobilization can be followed by measuring ON charge movement during a pulse and comparing it to OFF charge after the pulse. The OFF:ON ratio is near 1 for a pulse so short that no inactivation occurs, and the ratio drops to about one-third with a time course that parallels inactivation. Other correlations between inactivation and immobilization are that: (a) they have the same voltage dependence; (b) charge movement recovers with the time coures of recovery from inactivation. We interpret this to mean that the immobilized charge returns slowly to "off" position with the time course of recovery from inactivation, and that the small current generated is lost in base-line noise. At -150 mV recover is very rapid, and the immobilized charge forms a distinct slow component of current as it returns to off position. After destruction of inactivation by pronase, there is no immobilization of charge. A model is presented in which inactivation gains its voltage dependence by coupling to the activation gate.

scholarly journals Effects of Internal Divalent Cations on Voltage-Clamped Squid Axons

1974 ◽  
Vol 63 (6) ◽  
pp. 675-689 ◽  
Author(s):  
Ted Begenisich ◽  
Carl Lynch
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Divalent Cations ◽  
Sodium Current ◽  
Ionic Currents ◽  
Zinc Concentrations ◽  
Sodium Inactivation ◽  
Kinetics Of ◽  
Internal Calcium ◽  
Reversible Reduction

We have studied the effects of internally applied divalent cations on the ionic currents of voltage-clamped squid giant axons. Internal concentrations of calcium up to 10 mM have little, if any, effect on the time-course, voltage dependence, or magnitude of the ionic currents. This is inconsistent with the notion that an increase in the internal calcium concentration produced by an inward calcium movement with the action potential triggers sodium inactivation or potassium activation. Low internal zinc concentrations (∼1 mM) selectively and reversibly slow the kinetics of the potassium current and reduce peak sodium current by about 40% with little effect on the voltage dependence of the ionic currents. Higher concentrations (∼10 mM) produce a considerable (ca. 90%) nonspecific reversible reduction of the ionic currents. Large hyperpolarizing conditioning pulses reduce the zinc effect. Internal zinc also reversibly depolarizes the axon by 20–30 mV. The effects of internal cobalt, cadmium, and nickel are qualitatively similar to those of zinc: only calcium among the cations tested is without effect.

scholarly journals Kinetics of intramembrane charge movement and sodium current in frog node of Ranvier.

1982 ◽  
Vol 79 (4) ◽  
pp. 571-602 ◽  
Author(s):  
J M Dubois ◽  
M F Schneider
Keyword(s):  
Time Constant ◽  
Time Course ◽  
Sodium Current ◽  
Node Of Ranvier ◽  
Charge Movement ◽  
Intramembrane Charge Movement ◽  
Time Courses ◽  
Charge Movements ◽  
Kinetics Of ◽  
Microsecond Pulse

Intramembrane charge movement (Q) and sodium current (INa) were monitored in isolated voltage-clamped frog nodes of Ranvier, ON charge movements (QON) for pulses from the holding potential (-100 mV) to potentials V less than or equal to 0 mV followed single exponential time courses, whereas two exponentials were found for pulses to V greater than or equal to 20 mV. The voltage dependence of both QON and its time constant tauON indicated that the two ON components resolved at V greater than or equal to 20 mV were also present, though not resolvable, for pulses to V less than or equal to 0 mV. OFF charge movements (QOFF) monitored at various potentials were well described by single exponentials. When QOFF was monitored at -30 or -40 mV after a 200-microsecond pulse to +20 mV and QON was monitored at the same potential using pulses directly from -100 mV, tauON/tauOFF = 2.5 +/- 0.3. At a set OFF potential (-90 to -70 mV), tauOFF first increased with increasing duration tON of the preceding pulse to a given potential (0 to +30 mV) and then decreased with further increases in tON. The declining phase of tauOFF followed a time course similar to that of the decline in QOFF with tON. For the same pulse protocol, the OFF time constant tauNa for INA also first increased with tON but then remained constant over the tON interval during which tauOFF and QOFF were declining. After 200- or 300-microsecond pulses to +20, +20, or +50 mV, tauOFF/tauNa at -70 to -90 mV was 1.2 +/- 0.1. Similar tauOFF/tauNa ratios were predicted by channel models having three identical charged gating particles that can rapidly and reversibly form an immobile dimer or trimer after independently crossing the membrane from their OFF to their ON locations.

scholarly journals Phosphorylation modulates potassium conductance and gating current of perfused giant axons of squid.

1990 ◽  
Vol 95 (2) ◽  
pp. 245-271 ◽  
Author(s):  
C K Augustine ◽  
F Bezanilla
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Potassium Conductance ◽  
Charge Movement ◽  
Gating Current ◽  
Gating Currents ◽  
Internal Solution ◽  
Giant Axons ◽  
Gating Charge ◽  
Phosphorylation Reaction

The presence of internal Mg-ATP produced a number of changes in the K conductance of perfused giant axons of squid. For holding potentials between -40 and -50 mV, steady-state K conductance increased for depolarizations to potentials more positive than approximately -15 mV and decreased for smaller depolarizations. The voltage dependencies of both steady-state activation and inactivation also appears shifted toward more positive potentials. Gating kinetics were affected by internal ATP, with the activation time constant slowed and the characteristic delay in K conductance markedly enhanced. The rate of deactivation also was hastened during perfusion with ATP. Internal ATP affected potassium channel gating currents in similar ways. The voltage dependence of gating charge movement was shifted toward more positive potentials and the time constants of ON and OFF gating current also were slowed and hastened, respectively, in the presence of ATP. These effects of ATP on the K conductance occurred when no exogenous protein kinases were added to the internal solution and persisted even after removing ATP from the internal perfusate. Perfusion with a solution containing exogenous alkaline phosphatase reversed the effects of ATP. These results provide further evidence that the effects of ATP on the K conductance are a consequence of a phosphorylation reaction mediated by a kinase present and active in perfused axons. Phosphorylation appears to alter the K conductance of squid giant axons via a minimum of two mechanisms. First, the voltage dependence of gating parameters are shifted toward positive potentials. Second, there is an increase in the number of functional closed states and/or a decrease in the rates of transition between these states of the K channels.

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