Cadmium block of squid calcium currents. Macroscopic data and a kinetic model

The mechanism of Cd2+ block of Ca2+ currents (ICa) was explored in squid neurons using whole-cell patch clamp. Control currents activated sigmoidally, more rapidly at more positive potentials, and did not inactivate significantly. External Cd2+ up to 250 microM reduced ICa reversibly. For small depolarizations, the current for a step of 10 ms increased to a maintained value, resembling the control; but for Vm greater than 0 mV, the increase was followed by a decrease, as Cd2+ block became greater. Final block was greater for larger depolarizations. At 0 mV the half-blocking concentration was 125 microM. Tail currents, measured as channels close, had an initial "hook" when recorded in Cd2+: currents increased transiently, then decreased. This suggests that Cd2+ escapes from some channels, which then conduct briefly before closing. Analysis of tail currents shows that Cd2+ does not slow channel closing. The data can be explained if Cd2+ is a permeant blocker of Ca2+ channels and if channels can close when occupied by Cd2+. Cd2+ permeates the channels, but binds transiently to a site in the pore, obstructing the passage of other ions (e.g., Ca2+). Dwell time depends on the transmembrane potential, becoming shorter for more negative internal potentials. A five-state model was used to simulate the steady-state and kinetic features. It combines a Hodgkin-Huxley type m2 gating scheme and a one-site Woodhull ionic blockage model for a permeant blocker and includes a closed blocked state. To fit the data, the binding site for Cd2+ had to be near the outer end of the pore, with a well depth of -12.2 RT, and with a barrier at each end of the pore. The model predicts that the Cd2+ entry rate is nearly voltage independent, but the exit rate is steeply voltage dependent (e-fold/17 mV). Analysis further suggests that the channel closes at a normal rate with Cd2+ in the pore.     

L-type Ca2+ channels access multiple open states to produce two components of Bay K 8644-dependent current in GH3 cells

To determine the number of L-channel populations responsible for producing the two components of whole-cell L-type Ca2+ channel current revealed by Bay K 8644 (Fass, D.M., and E.S. Levitan. 1996. J. Gen. Physiol. 108:1-11), L-type Ca2+ channel activity was recorded in cell- attached patches. Ensemble tail currents from most (six out of nine) single-channel patches had double-exponential time courses, with time constants that were similar to whole-cell tail current decay values. Also, in single-channel patches subjected to two different levels of depolarization, ensemble tail currents exactly reproduced the voltage dependence of activation of the two whole-cell components: The slow component is activated at more negative potentials than the fast component. In addition, deactivation of Bay K 8644-modified whole-cell L-current was slower after long (100-ms) depolarizations than after short (20-ms) depolarizations, and this phenomenon was also evident in ensemble tail currents from single L-channels. Thus, a single population of L-channels can produce the two components of macroscopic L-current deactivation. To determine how individual L-channels produce multiple macroscopic tail current components, we constructed ensemble tail currents from traces that contained a single opening upon repolarization and no reopenings. These ensemble tails were biexponential. This type of analysis also revealed that reopenings do not contribute to the slowing of tail current deactivation after long depolarizations. Thus, individual L-channels must have access to several open states to produce multiple macroscopic current components. We also obtained evidence that access to these open states can vary over time. Use of several open states may give L-channels the flexibility to participate in many cell functions.     

Isolation of myocardial L-type calcium channel gating currents with the spider toxin omega-Aga-IIIA

The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) blocks ionic current through L-type Ca channels in guinea pig atrial cells without affecting the associated gating currents. omega-Aga-IIIA permits the study of L- type Ca channel ionic and gating currents under nearly identical ionic conditions. Under conditions that isolate L-type Ca channel currents, omega-Aga-IIIA blocks all ionic current during a test pulse and after repolarization. This block reveals intramembrane charge movements of equal magnitude and opposite sign at the beginning of the pulse (Q(on)) and after repolarization (Q(off)). Q(on) and Q(off) are suppressed by 1 microM felodipine, saturate with increasing test potential, and are insensitive to Cd. The decay of the transient current associated with Q(on) is composed of fast and slow exponential components. The slow component has a time constant similar to that for activation of L-type Ca channel ionic current, over a broad voltage range. The current associated with Q(off) decays monoexponentially and more slowly than ionic current. Similar charge movements are found in guinea pig tracheal myocytes, which lack Na channels and T-type Ca channels. The kinetic and pharmacological properties of Q(on) and Q(off) indicate that they reflect gating currents associated with L-type Ca channels. omega-Aga-IIIA has no effect on gating currents when ionic current is eliminated by stepping to the reversal potential for Ca or by Cd block. Gating currents constitute a significant component of total current when physiological concentrations of Ca are present and they obscure the activation and deactivation of L-type Ca channels. By using omega- Aga-IIIA, we resolve the entire time course of L-type Ca channel ionic and gating currents. We also show that L- and T-type Ca channel ionic currents can be accurately quantified by tail current analysis once gating currents are taken into account.     

Fast-deactivating calcium channels in chick sensory neurons

Whole-cell Ca and Ba currents were studied in chick dorsal root ganglion (DRG) cells kept 6-10 in culture. Voltage steps with a 15-microseconds rise time were imposed on the membrane using an improved patch-clamp circuit. Changes in membrane current could be measured 30 microseconds after the initiation of the test pulse. Currents through Ca channels were recorded under conditions that eliminate Na and K currents. Tail currents, associated with Ca channel closing, decayed in two distinct phases that were very well fitted by the sum of two exponentials. The time constants tau f and tau s were near 160 microseconds and 1.5 ms at -80 mV, 20 degrees C. The tail current components, called FD and SD (fast-deactivating and slowly deactivating), are Ca channel currents. They were greatly reduced when Mg2+ replaced all other divalent cations in the bath. The SD component inactivated almost completely as the test pulse duration was increased to 100 ms. It was suppressed when the cell was held at membrane potentials positive to -50 mV and was blocked by 100-200 microM Ni2+. This behavior indicates that the SD component was due to the closing of the low-voltage-activated (LVA) Ca channels previously described in this preparation. The FD component was fully activated with 10-ms test pulses to +20 mV at 20 degrees C, and inactivated to approximately 30% during 500-ms test pulses. It was reduced in amplitude by holding at -40 mV, but was only slightly reduced by micromolar concentrations of Ni2+. Replacement of Ca2+ with Ba2+ increased the FD tail current amplitudes by a factor of approximately 1.5. The deactivation kinetics did not change (a) as channels inactivated during progressively longer pulses or (b) when the degree of activation was varied. Further, tau f was affected neither by changing the holding potential nor by varying the test pulse amplitude. Lowering the temperature from 20 to 10 degrees C decreased tau f by a factor of 2.5. In all cases, the FD component was very well fitted by a single exponential. There was no indication of an additional tail component of significant size. Our findings indicate that the FD component is due to closing of a single class of Ca channels that coexist with the LVA Ca channel type in chick DRG neurons.     

State-independent block of BK channels by an intracellular quaternary ammonium

Intracellular blockade by quaternary ammonium (QA) molecules of many potassium channels is state dependent, where the requirement for channel opening is evidenced by a time-dependent component of block in the macroscopic record. Whether this is the case for Ca(2+)- and voltage-activated potassium (BK) channels, however, remains unclear. Previous work (Li, W., and R.W. Aldrich. 2004. J. Gen. Physiol. 124:43-57) tentatively proposed a state-dependent, trapping model, but left open the possibility of state-independent block. Here, we found BK channel blockade by a novel QA derivative, bbTBA, was time dependent, raising the possibility of state-dependent, open channel block. Alternatively, the observed voltage dependence of block could be sufficient to explain time-dependent block. We have used steady-state and kinetic measurements of bbTBA blockade in order to discriminate between these two possibilities. bbTBA did not significantly slow deactivation kinetics at potentials between -200 and -100 mV, suggesting that channels can close unhindered by bound bbTBA. We further find no evidence that bbTBA is trapped inside BK channels after closing. Measurements of steady state fractional block at +40 mV revealed a 1.3-fold change in apparent affinity for a 33-fold change in P(o), in striking contrast to the 31-fold change predicted by state-dependent block. Finally, the appearance of a third kinetic component of bbTBA blockade at high concentrations is incompatible with state-dependent block. Our results suggest that access of intracellular bbTBA to the BK channel cavity is not strictly gated by channel opening and closing, and imply that the permeation gate for BK channels may not be intracellular.     

Mechanism of Ba2+ block of M-like K channels of rod photoreceptors of tiger salamanders

IKx is a voltage-dependent K+ current in the inner segment of rod photoreceptors that shows many similarities to M-current. The depression of IKx by external Ba2+ was studied with whole-cell voltage clamp. Ba2+ reduced the conductance and voltage sensitivity of IKx tail currents and shifted the voltage range over which they appeared to more positive potentials. These effects showed different sensitivities to Ba2+: conductance was the least sensitive (K0.5 = 7.6 mM), voltage dependence intermediate (K0.5 = 2.4 mM) and voltage sensitivity the most sensitive (K0.5 = 0.2 mM). Ca2+, Co2+, Mn2+, Sr2+, and Zn2+ did not have actions comparable to Ba2+ on the voltage dependence or the voltage sensitivity of IKx tail currents. In high K+ (100 mM), the voltage range of activation of IKx was shifted 20 mV negative, as was the tau-voltage relation. High K+ did not prevent the effect of Ba2+ on conductance, but abolished its ability to affect voltage dependence and voltage sensitivity. Ba2+ also altered the apparent time-course of activation and deactivation of IKx. Low Ba2+ (0.2 mM) slowed both deactivation and activation, with most effect on deactivation; at higher concentrations (1-25 mM), deactivation and activation time courses were equally affected, and at the highest concentrations, 5 and 25 mM Ba2+, the time course became faster than control. Rapid application of 5 mM Ba2+ suggested that the time dependent currents in Ba2+ reflect in part the slow voltage-dependent block and unblock of IKx channels by Ba2+. This blocking action of Ba2+ was steeply voltage- dependent with an apparent electrical distance of 1.07. Ba2+ appears to interact with IKx channels at multiple sites. A model which assumes that Ba2+ has a voltage-independent and a voltage-dependent blocking action on open or closed IKx channels reproduced many aspects of the data; the voltage-dependent component could account for both the Ba(2+)- induced shift in voltage dependence and reduction in voltage sensitivity of IKx tail currents.     

Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon.

Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.     

Interaction of Ca2+ agonist and depolarization on Ca2+ channel current in guinea pig detrusor cells [published erratum appears in J Gen Physiiol 1996 Jun;107(6):755]

The interaction of large depolarization and dihydropyridine Ca2+ agonists, both of which are known to enhance L-type Ca2+ channel current, was examined using a conventional whole-cell clamp technique. In guinea pig detrusor cells, only L-type Ca2+ channels occur. A second open state (long open state: O2) of the Ca2+ channels develops during large depolarization (at +80 mV, without Ca2+ agonists). This was judged from lack of inactivation of the Ca2+ channel current during the large depolarizing steps (5 s) and slowly deactivating inward tail currents (= 10-15 ms) upon repolarization of the cell membrane to the holding potential (-60 mV). Application of Bay K 8644 (in 2.4 mM Ca(2+)- containing solutions) increased the amplitude of the Ca2+ currents evoked by simple depolarizations, and made it possible to observe inward tail currents (= 2.5-5 ms at -60 mV). The open state induced by large depolarization (O2*) in the Bay K 8644 also seemed hardly to inactivate. After preconditioning with large depolarizing steps, the decay time course of the inward tail currents upon repolarization to the holding potential (-60 mV) was significantly slowed, and could be fitted reasonably with two exponentials. The fast and slow time constants were 10 and 45 ms, respectively, after 2 s preconditioning depolarizations. Qualitatively the same results were obtained using Ba2+ as a charge carrier. Although the amplitudes of the inward currents observed in the test step and the subsequent repolarization to the holding potential were decreased in the same manner by additional application of nifedipine (in the presence of Bay K 8644), the very slow deactivation time course of the tail current was little changed. The additive enhancement by large depolarization and Ca2+ agonists of the inward tail current implies that two mechanisms separately induce long opening of the Ca2+ channels: i.e., that there are four open states.     

Slow deactivation and U-shaped inactivation properties in cloned Cav1.2b channels in Chinese hamster ovary cells

Whole-cell patch-clamp techniques were applied to Chinese hamster ovary cells stably expressing cloned smooth muscle Ca(2+) channel alpha(1)-subunits. In the presence of Ba(2+) as a charge carrier, U-shaped inactivation was observed in the presence and absence of Ca(2+) agonists. Also, tail currents deactivated slowly when conditioning steps of positive potential were applied. The deactivation time constant was decreased by hyperpolarizing the repolarization step. Application of ATP-gamma-S or H-7 had little effect on the conditions necessary to induce slow tail, suggesting involvement of physical processes in the channel protein. In the presence of Bay K 8644, additional application of nifedipine decreased the amplitudes of the test and tail currents induced by a test step preceded by a conditioning step to +80 mV, but did not affect the decay time constant of the tail current. From these results and assumptions we have drawn up a kinetic scheme with one closed state, two open states (O(1), O(2)) and two inactivated states linked to the closed state and open state O(1), respectively, i.e., open state O(2) protected from inactivation. Computer calculation reconstructed slow deactivation and U-shaped inactivation properties. A similar kinetic scheme with Ca(2+)-agonist-binding states accounted for the results in the presence of Ca(2+) agonists.     

Action potential waveforms reveal simultaneous changes in ICa and IK produced by 5-HT in rat dorsal raphe neurons.

Action potentials were recorded from serotonergic dorsal raphe (DR) neurons acutely isolated from the adult rat brain. Action potential waveforms were used as command potentials for whole-cell patch-clamp studies to investigate the Ca2+ and K+ currents underlying action potentials and the modulatory effects of 5-Hydroxytryptamine (5-HT) on them. These data were compared with currents elicited by using rectangular voltage steps of the type commonly used in voltage-clamp experiments. In the same cell, 5-HT simultaneously augmented K+ currents and inhibited Ca2+ currents. Experimental conditions were chosen which allowed us to examine the action of 5-HT on K+ and Ca2+ currents simultaneously or in isolation; 5-HT produced a larger inhibition of calcium current during an action potential waveform compared with that measured by using rectangular steps of voltage. A possible explanation for this finding is that the maximal inhibition is seen immediately after a voltage jump and then decreases with time. Action potentials are, in general, so brief that little time-dependent relief of block is observed. Most of the inhibition of Ca2+ current resulted from a direct effect on Ca2+ channels rather than a shortening of the action potential. The inhibition of Ca2+ current by 5-HT also decreased the Ca(2+)-activated K+ currents. These results suggest that 5-HT reduces DR neuron excitability by the simultaneous activation of K+ channel currents open at the resting potential and the suppression of Ca2+ channel currents.     

Block by internal Mg2+ causes voltage-dependent inactivation of Kv1.5

Internal Mg2+ blocks many potassium channels including Kv1.5. Here, we show that internal Mg2+ block of Kv1.5 induces voltage-dependent current decay at strongly depolarised potentials that contains a component due to acceleration of C-type inactivation after pore block. The voltage-dependent current decay was fitted to a bi-exponential function (tau(fast) and tau(slow)). Without Mg2+, tau(fast) and tau(slow) were voltage-independent, but with 10 mM Mg2+, tau(fast) decreased from 156 ms at +40 mV to 5 ms at +140 mV and tau(slow) decreased from 2.3 s to 206 ms. With Mg2+, tail currents after short pulses that allowed only the fast phase of decay showed a rising phase that reflected voltage-dependent unbinding. This suggested that the fast phase of voltage-dependent current decay was due to Mg2+ pore block. In contrast, tail currents after longer pulses that allowed the slow phase of decay were reduced to almost zero suggesting that the slow phase was due to channel inactivation. Consistent with this, the mutation R487V (equivalent to T449V in Shaker) or increasing external K+, both of which reduce C-type inactivation, prevented the slow phase of decay. These results are consistent with voltage-dependent open-channel block of Kv1.5 by internal Mg2+ that subsequently induces C-type inactivation by restricting K+ filling of the selectivity filter from the internal solution.     

Mechanism of inactivation gating of human T-type (low-voltage activated) calcium channels

Recovery from inactivation of T-type Ca channels is slow and saturates at moderate hyperpolarizing voltage steps compared with Na channels. To explore this unique kinetic pattern we measured gating and ionic currents in two closely related isoforms of T-type Ca channels. Gating current recovers from inactivation much faster than ionic current, and recovery from inactivation is much more voltage dependent for gating current than for ionic current. There is a lag in the onset of gating current recovery at -80 mV, but no lag is discernible at -120 mV. The delay in recovery from inactivation of ionic current is much more evident at all voltages. The time constant for the decay of off gating current is very similar to the time constant of deactivation of open channels (ionic tail current), and both are strongly voltage dependent over a wide voltage range. Apparently, the development of inactivation has little influence on the initial deactivation step. These results suggest that movement of gating charge occurs for inactivated states very quickly. In contrast, the transitions from inactivated to available states are orders of magnitude slower, not voltage dependent, and are rate limiting for ionic recovery. These findings support a deactivation-first path for T-type Ca channel recovery from inactivation. We have integrated these concepts into an eight-state kinetic model, which can account for the major characteristics of T-type Ca channel inactivation.     

Ca2+ currents in cerebral artery smooth muscle cells of rat at physiological Ca2+ concentrations

Single Ca2+ channel and whole cell currents were measured in smooth muscle cells dissociated from resistance-sized (100-microns diameter) rat cerebral arteries. We sought to quantify the magnitude of Ca2+ channel currents and activity under the putative physiological conditions of these cells: 2 mM [Ca2+]o, steady depolarizations to potentials between -50 and -20 mV, and (where possible) without extrinsic channel agonists. Single Ca2+ channel conductance was measured over a broad range of Ca2+ concentrations (0.5-80 mM). The saturating conductance ranged from 1.5 pS at 0.5 mM to 7.8 pS at 80 mM, with a value of 3.5 pS at 2 mM Ca (unitary currents of 0.18 pA at -40 mV). Both single channel and whole cell Ca2+ currents were measured during pulses and at steady holding potentials. Ca2+ channel open probability and the lower limit for the total number of channels per cell were estimated by dividing the whole-cell Ca2+ currents by the single channel current. We estimate that an average cell has at least 5,000 functional channels with open probabilities of 3.4 x 10(-4) and 2 x 10(-3) at -40 and -20 mV, respectively. An average of 1-10 (-40 mV and -20 mV, respectively) Ca2+ channels are thus open at physiological potentials, carrying approximately 0.5 pA steady Ca2+ current at -30 mV. We also observed a very slow reduction in open probability during steady test potentials when compared with peak pulse responses. This 4- 10-fold reduction in activity could not be accounted for by the channel's normal inactivation at our recording potentials between -50 and -20 mV, implying that an additional slow inactivation process may be important in regulating Ca2+ channel activity during steady depolarization.     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

Patch and whole cell calcium currents recorded simultaneously in snail neurons

The flow of Ca ions through single Ca channels has been examined. The gigaseal method was used on identifiable snail neurons that were voltage clamped using a two-microelectrode voltage clamp method. Average Ca patch currents and whole cell currents have similar time courses. They are affected similarly by changes in temperature. The differences in amplitude and inactivation between Ba and Ca whole cell currents were present in the patch records. The stationary noise spectra recorded from ensembles of multichannel patches have two components with fast and slow time constants equivalent to two components in the whole cell tail current relaxations. Elementary current amplitudes measured from the variance-mean relationship and from noise spectra gave values comparable to measurements from single channels. The single channel I-V relationship was curvilinear and the maximum slope conductance in 40 mM Cao was 7 pS. The amplitude of unitary currents was unchanged at long times when inactivation had occurred; hence depletion is not involved in this process. Channel density was approximately 3 microns-2 and was the same for Ba and Ca currents. The whole cell asymmetry currents gave very large values for the gating charge per channel. Changes in temperature from 29 to 9 degrees C had only a slight effect on the two Ca tail current tau's at potentials where turn-on of patch and whole cell currents was markedly slowed and the peak amplitudes were reduced by one-third. Single channel recordings were obtained at these two temperatures, and the mean open time and the fast component of the closed times were scarcely affected. Unit amplitudes were reduced by 30% and the slow closed time component was doubled. Therefore, peak currents and the slow closed time component was doubled. Therefore, peak currents were reduced partly as a result of the reduction in unit amplitude, but mainly as a result of a reduction in opening probability, the latter arising from an increase of the long closed times. It is concluded that the behavior of single Ca channels in membrane patches is the same as it is in whole cells. Cooling from 29 to 9 degrees C acts primarily on transitions among closed states and has little effect on the open to closed transition.     

Statistical analysis of single sodium channels. Effects of N-bromoacetamide

Currents were obtained from single sodium channels in outside-out excised patches of membrane from the cell line GH3. The currents were examined in control patches and in patches treated with N- bromoacetamide ( NBA ) to remove inactivation. The single-channel current-voltage relationship was linear over the range -60 to + 10 mV, and was unaffected by NBA . The slope conductance at 9.3 degrees C was 12 pS, and the Q10 for single channel currents was about 1.35. The currents in both control and NBA -treated patches showed evidence of a slow process similar to desensitization in acetylcholine-receptor channels. This process was especially apparent at rapid rates of stimulation (5 Hz), where openings occurred in clusters of records. The clustering of records with and without openings was analyzed by runs analysis, which showed a statistically significant trend toward nonrandom ordering in the responses of channels to voltage pulses. NBA made this nonrandom pattern more apparent. The probability that an individual channel was "hibernating" during an activating depolarization was estimated by a maximum likelihood method. The lifetime of the open state was also estimated by a maximum likelihood method, and was examined as a function of voltage. In control patches the open time was mildly voltage-dependent, showing a maximum at about -50 mV. In NBA -treated patches the open time was greater than in the control case and increased monotonically with depolarization; it asymptotically approached that of the control patches at hyperpolarized potentials. By comparing channel open times in control and NBA -treated patches, we determined beta A and beta I, the rate constants for closing activation gates and fast inactivation gates. Beta I was an exponential function of voltage, increasing e-fold for 34 mV. beta A had the opposite voltage dependence. The probability of an open channel closing its fast inactivation gate, rather than its activation gate, increased linearly with depolarization from -60 to -10 mV. These results indicate that inactivation is inherently voltage dependent.     

ATP-Dependent Regulation of an Anion Channel at the Plasma Membrane of Protoplasts from Epidermal Cells of Arabidopsis Hypocotyls

Although Arabidopsis is the object of many genetic and molecular biology investigations, relatively few studies deal with regulation of its transmembrane ion exchanges. To clarify the role of ion transport in plant development, organ-and tissue-specific ion channels must be studied. We identified a voltage-dependent anion channel in epidermal cells of Arabidopsis hypocotyls, thus providing a new example of the occurrence of voltage-dependent anion channels in a specific plant cell type distinct from the stomatal guard cell. The Arabidopsis hypocotyl anion channel is able to function under two modes characterized by different voltage dependences and different kinetic behaviors. This switch between a fast and a slow mode is controlled by ATP. In the presence of intracellular ATP (fast mode), the channels are closed at resting potentials, and whole-cell currents activate upon depolarization. After activation, the anion current deactivates rapidly and more and more completely at potentials negative to the peak. In the absence of ATP, the current switches from this fast mode to a mode characterized by a slow and incomplete deactivation at resting potentials. In addition, the whole-cell currents can be correlated with the activity of single channels. In the outside-out configuration, the presence of ATP modulates the mean lifetimes of the open and closed states of the channel at hyperpolarized potentials, thus controlling its open probability. The fact that ATP-dependent voltage regulation was observed in both whole-cell and outside-out configurations suggests that a single type of anion channel can switch between two modes with distinct functional properties.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.     

Kinetics of interaction of disopyramide with the cardiac sodium channel: Fast dissociation from open channels at normal rest potentials

Block of cardiac sodium channels is enhanced by repetitive depolarization. It is not clear whether the changes in drug binding result from a change in affinity that is dependent on voltage or on the actual state of the channel. This question was examined in rabbit ventricular myocytes by analyzing the kinetics of block of single sodium channel currents with normal gating kinetics or channels with inactivation and deactivation slowed by pyrethrin toxins. At −20 and −40 mV, disopyramide 100 μm blocked the unmodified channel. Mean open time decreased45 and34% at −20 and −40 mV during exposure to disopyramide. Exposure of cells to the pyrethrin toxins deltamethrin or fenvalrate caused at least a tenfold increase in mean open time, and prominent tail currents could be recorded at the normal resting potential. The association rate constant of disopyramide for the normal and modified channel at −20 mV was similar, ∼10×10⁶/m/sec. During exposure to disopyramide, changes in open and closed times and in open channel noise at −80 and −100 mV are consistent with fast block and unblocking events at these potentials. This contrasts with the slow unbinding of drug from resting channels at similar potentials. We conclude that the sodium channel state is a critical determinant of drug binding and unbinding kinetics.