Selective Inhibition of Transient K+ Current by La3+ in Crab Peptide-Secretory Neurons

Shumin Duan and Ian M. Cooke

Békésy Laboratory of Neurobiology and Department of Zoology, University of Hawaii, Honolulu, Hawaii 96822


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Duan, Shumin and Ian M. Cooke. Selective inhibition of transient K+ current by La3+ in crab peptide-secretory neurons. Although divalent cations and lanthides are well-known inhibitors of voltage-dependent Ca2+ currents (ICa), their ability to selectively inhibit a voltage-gated K+ current is less widely documented. We report that La3+ inhibits the transient K+ current (IA) of crab (Cardisoma carnifex) neurosecretory cells at ED50 ~5 µM, similar to that blocking ICa, without effecting the delayed rectifier K+ current (IK). Neurons were dissociated from the major crustacean neuroendocrine system, the X-organ-sinus gland, plated in defined medium, and recorded by whole cell patch clamp after 1-2 days in culture. The bath saline included 0.5 µM TTX and 0.5 mM CdCl2 to eliminate inward currents. Responses to depolarizing steps from a holding potential of -40 mV represented primarily IK. They were unchanged by La3+ up to 500 µM. Currents from -80 mV in the presence of 20 mM TEA were shown to represent primarily IA. La3+ (with TEA) reduced IA and maximum conductance (GA) by ~10% for 1 µM and another 10% each in 10 and 100 µM La3+. Normalized GA-V curves were well fit with a single Boltzmann function, with V1/2 +4 mV and slope 15 mV in control; V1/2 was successively ~15 mV depolarized and slope increased ~2 mV for each of these La3+ concentrations. Cd2+ (1 mM), Zn2+ (200 µM), and Pb2+ (100 µM) or removal of saline Mg2+ (26 mM) had little or no effect on IA. Steady-state inactivation showed similar right shifts (from V1/2 -39 mV) and slope increases (from 2.5 mV) in 10 and 100 µM La3+. Time to peak IA was slowed in 10 and 100 µM La3+, whereas curves of normalized time constants of initial decay from peak IA versus Vc were right-shifted successively ~15 mV for the three La3+ concentrations. The observations were fitted by a Woodhull-type model postulating a La3+-selective site that lies 0.26-0.34 of the distance across the membrane electric field, and both block of K+ movement and interaction with voltage-gating mechanisms; block can be relieved by depolarization and/or outward current. The observation of selective inhibition of IA by micromolar La3+ raises concerns about its use in studies of ICa to evaluate contamination by outward current.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lanthanum chloride is widely used as a potent blocker of Ca2+ permeation through membranes. It is effective at micromolar concentrations when added to physiological salines having their usual concentrations of divalent cations. It is used to completely block calcium currents (ICa) to evaluate whether any voltage-dependent outward currents contaminate the currents recorded under regimes designed to isolate ICa and thus improve the characterization of voltage-dependent ICa. We found that La3+ inhibits the voltage-dependent, rapidly inactivating potassium current (IA) of crab secretory neurons at concentrations similar to those inhibiting ICa without affecting the delayed rectifier potassium current (IK).

Although inhibitory effects of divalent Ca2+-channel blockers on both transient and delayed outward currents were described for a variety of invertebrate (e.g., Thompson 1977) and vertebrate neurons (e.g., Mayer and Sugiyama 1988), there appear to be few reports of selective effects of La3+ on IA at concentrations normally used to block ICa. Because negative results are rarely reported, it is difficult to know how widely the effects of low concentrations of La3+ on potassium currents have been examined. Selective effects of La3+ on the IA component of outward current of rat cerebellar granule cells (Watkins and Mathie 1994) and hippocampal neurons (Talukder and Harrison 1995) point to the possibility that such actions may be more general than is currently appreciated. Except for an abstract (Duan and Cooke 1997), the effects of La3+ on potassium currents of crustacean neurons have not to our knowledge been previously reported.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dissection and culturing

The procedures used to dissociate and culture X-organ neurons from the semiterrestrial tropical crab, Cardisoma carnifex Herbst, were described in detail elsewhere (Cooke et al. 1989; Grau and Cooke 1992). Briefly, the X-organ with <1 mm of the axon tract was removed from the eye stalk of adult male crabs and agitated in the dark for 1.5 h in a Ca2+/Mg2+-free saline containing 0.1% trypsin (Gibco). A large volume of Ca2+/Mg2+-free saline was then added to retard enzymatic activity, and the cells were dissociated by gentle trituration in a 60-µl drop of culture medium on 35-mm Primaria dishes (Falcon 3801). The dishes were carefully flooded after allowing 1-2 h for the cells to adhere to the substratum. The culture medium consisted of Leibowitz L-15 (Gibco) diluted 1:1 with double-strength crab saline to which D-glucose (120 mM, Fisher), L-glutamine (2 mM, Sigma), and gentamicin (50 mg/ml, Gibco) were added. Cultures were maintained in humidified incubators (Billups-Rothenberg) in the dark at 22-24°C.

Experiments were performed on cultured X-organ somata that were 2-3 days in culture. Cells whose regenerative outgrowth took the form of large, lamellipodial growth cones ("veilers") (Grau and Cooke 1992) and were thus identifiable as containing crustacean hyperglycemic hormone (Keller et al. 1995) were chosen for this study. Before starting electrophysiological recording, the culture dish was rinsed three times, and the medium was replaced with filtered crab saline consisting of (in mM) 440 NaCl, 11 KCl, 13.3 CaCl2, 26 MgCl2, 26 Na2SO4, and 10 HEPES, pH 7.4 with NaOH. During the experiments, the dish was constantly superfused with crab saline at a rate of ~0.2 ml/min. A self-priming siphon maintained a relatively constant fluid level. Somata were viewed with a Nikon Diaphot inverted microscope with a ×40 objective and Hoffman modulation optics.

La3+ application

The use of a miniature Y-tube (Murase et al. 1989) manipulated to within <25 µm of the soma permitted rapid (<100 ms) application of La3+-containing saline to the neuron with minimal dilution or mixing during patch clamping. The solutions to be applied, held in reservoirs higher than the recording bath, are drawn through the arms of the Y, which are of larger diameter tubing (PE 50) than the stem (PE10), by gentle suction; some bath fluid is drawn in through the stem ensuring that no agent is applied unintentionally. A solenoid pinch valve controlled by a Grass S-15 stimulator through which the arm on the suction side of the Y passes is used to stop the suction for a selected duration, permitting gravity flow of the material out of the Y while suction is blocked.

Electrophysiology

Voltage-clamp recordings were obtained in the whole cell patch-clamp configuration with an EPC9 amplifier (Instrutech, NY). Data acquisition, storage, and analysis were performed with HEKA software (Instrutech, NY) run on a Macintosh Centris 650 computer. Correction for leak was achieved with a p/N of four scaled subtractions obtained at zero-current hyperpolarizing command potentials. Signals were filtered with a corner frequency of 2.9 kHz. All experiments were recorded at room temperature (22-26°C). Pipettes used to obtain tight-seal whole cell recordings were pulled from Kimax thin-walled glass capillaries (1.5-1.8 mm OD) on a vertical puller (David Kopf Instruments, TW 150F-4). Pipettes were coated with dental wax to reduce capacitance and fire polished with a microforge (Narishige, model MF-83). Pipettes filled with the intracellular solution and immersed in the bath had resistances ranging from 1.5 to 5 MOmega but typically 1.5 to 3 MOmega . The intracellular solution, unless otherwise noted, was (in mM) 300 KCl, 10 NaCl, 5 Mg-ATP, 5 BAPTA, and 50 HEPES, pH adjusted to 7.4 with KOH. The extracellular solution was crab saline as described previously with 120 mM D-glucose and with 0.5 µM TTX and 0.5 mM CdCl2 to suppress inward currents. The tonicity was adjusted with sucrose to 1,095-1,100 mosm for intracellular solutions and 1,100 mosm for extracellular solutions. After establishing an electrode seal and breaking in, neurons were held at -50 mV, a value close to the usual resting potential and therefore requiring very little current, when recordings were not being made. Series resistance (Rs), was <6 MOmega , typically ~3 MOmega ; it was compensated to the maximum possible without causing oscillation, ringing. or overshoot. Larger cells (capacitance >50 pF) could be more completely compensated and were compensated by 85-90%. The few smaller cells recorded could be less fully compensated but were never compensated at <60%. These cells also had smaller currents. The data were not corrected for uncompensated Rs; we estimate that errors in reported Vm caused by Rs are <5 mV. To record voltage-gated currents the potential was stepped from -50 mV to the intended holding potential (Vh) for 300 ms before initiating additional voltage commands.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of IA and IK

In this study, outward currents were examined with a bathing saline containing 0.5 µM TTX and 0.5-1 mM Cd2+ to eliminate possible competing inward Na+ and Ca2+ currents (Meyers et al. 1992; Richmond et al. 1995). The presence of TTX increased the size of the initial outward current, whereas addition of Cd2+ to TTX-containing saline decreased a dip in the current traces between the early peak and later outward current but did not alter the peak (<15 ms); the amplitude of the late outward current (measured at 90 ms) was reduced by the presence of Cd2+ but by <10% (data not shown) as also noted by Meyers et al. (1992).

As previously described for the cultured C. carnifex X-organ neurons (Meyers et al. 1992), outward K+ currents having typical characteristics of IA and IK can be readily isolated. Currents representing a Ca2+-activated potassium current were not resolved and if present were very small. The characteristics and current densities of IA and IK did not differ systematically among X-organ neurons having different morphologies of outgrowth in culture nor with time in culture (Meyers et al. 1992). Exploiting the rapid inactivation of IA during depolarization, currents in response to incremented depolarizing voltage commands were recorded from a holding potential (Vh) of -80 mV, at which IA is fully available for activation (Fig. 1A), and again from Vh -40 mV (Fig. 1B), at which IA is almost fully inactivated, leaving responses primarily representing IK. The responses at corresponding commands from Vh -40 mV were subtracted from those at -80 mV to provide estimates of IA (Fig. 1E).



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Fig. 1. Separation of outward currents of crab peptidergic neurons in culture. For this and all subsequent figures, Cardisoma carnifex X-organ neurons, dissociated and cultured 2-3 days in defined medium, showing a lamellipodial growth cone, were recorded under whole cell patch clamp applied to the soma in crab saline containing 0.5 µM TTX and 0.5-1 mM Cd2+ to block INa and ICa. Each panel shows superimposed current responses, capacitance, and leak corrected and series resistance compensated >60% to voltage steps from -50 to +40 mV, in 15-mV increments, from the holding potential (Vh) indicated. All records are from the same neuron. A: responses to depolarizing commands from Vh -80 mV show a fast, initial peak outward current (IA), which decays and is followed by sustained current (IK). B: responses from Vh -40 mV show little initial transient current consistent with inactivation of IA, leaving primarily IK. C: responses from Vh -80 mV in the presence of 4-AP, a selective inhibitor of IA; comparison with B shows the remaining IK is similar for Vh -80 and -40 mV. D: in the presence of TEA, a selective inhibitor of IK, transient peaks remain (compare with A), but late current is reduced. E: current remaining in TEA (D) closely resembles current calculated by subtraction of the records of B from A. Except in Fig. 2, subsequent records were obtained from Vh -80 mV in 20 mM TEA except as noted and are considered to represent predominantly IA.

Because La3+ causes voltage shifts of IA activation and inactivation as detailed subsequently, the use of different holding potentials to isolate the currents was impractical. We thus evaluated the effectiveness, compared with the use of different Vh, of using pharmacological blockers to separate IK and IA (Thompson 1977). As for other neurons, 4-aminopyridine (4-AP, 5 mM) effectively and relatively selectively blocked IA (Fig. 1C), whereas TEA (20 mM in the bath) selectively blocked IK (Fig. 1D) and revealed transient outward currents closely matching those obtained by subtraction (Fig. 1E). The slightly smaller currents seen in Fig. 1E compared with Fig. 1D reflect the presence of IA remaining in responses from Vh -40 mV (Fig. 1B). These can be evaluated by comparing Fig. 1B, in which a small initial hump is present in the records, with Fig. 1C in which 4-AP has more completely blocked IA.

It will be noted that the currents we attribute as primarily representing IA, whether observed as the result of subtraction of currents obtained from Vh -40 from those at Vh -80 mV or from Vh -80 in the presence of 20 mM TEA, do not completely inactivate over the duration of depolarizations lasting >100 ms. Whether this residual current, consistently amounting to ~20% of peak IA, represents ion movement through channels responsible for IA or for IK remains undetermined. We have not corrected measurements of IA for this noninactivating component of current.

Unless otherwise noted, the observations on outward current presented subsequently were obtained in the presence of 0.5 µM TTX, 0.5-1 mM Cd2+, and 20 mM TEA. Under these conditions inward current was not apparent; voltage-dependent outward current will be referred to as IA.

La3+ inhibits IA but not IK

Figure 2 shows records of outward currents in saline with TTX and Cd2+, but without TEA (A) and after the addition of 100 µM La3+ (B) recorded from Vh -80 mV and therefore eliciting both IA and IK (left panels) and from Vh -40, primarily IK (right panels). A neuron showing relatively large IK was selected for this example. The early peak evoked from Vh -80 mV representing IA is absent in La3+, whereas the late and sustained currents (here measured at ~90 ms) are unaffected. In Fig. 2C, the measurements of current at 90 ms for control saline from Vh -40 mV are plotted with the observations in La3+ from both Vh -40 and -80 mV. The responses are almost superimposable. Concentrations of La3+ >0.5 mM were required to obtain a noticeable effect on IK, seen as a right-shift of the current-voltage relation (data not shown).



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Fig. 2. La3+ does not affect IK. A: outward currents recorded as in Fig. 1 (saline with 0.5 µM TTX and 0.5 mM Cd2+, but without TEA). B: records from the same neuron after addition of 100 µM La3+. Initial transient (IA) present in control records from Vh -80 mV is absent. C: I-V plot of late current (IK) measured at ~90 ms. Amplitudes of IK in control records from Vh -40 and those in the presence of La3+ from both Vh -40 and -80 mV are indistinguishable. This neuron was chosen for its large IK (note reduced scale relative to Fig. 1).

Figure 3 presents typical observations on IA current (isolated by recording in saline with TTX, Cd2+, and TEA) from a veiling neuron exposed to a wide range of La3+ concentrations. In Fig. 3A, currents recorded in response to steps to +15 mV from Vh -80 mV in control saline (a command evoking ~80% of the maximum response) and saline with 1, 10, and 100 µM La3+ are superimposed. The initial outward current peak is reduced, and its activation and inactivation slowed with increasing [La3+]; in 100 µM no peak is seen in response to the +15 mV command, and there is only a non- or slowly inactivating residual current. With more depolarizing commands peaks are observed, as reflected in plots of the conductance giving rise to IA, GA versus Vc (Fig. 3, B and C). As mentioned previously, the responses in La3+ at modest Vc lacking an initial peak may represent IK that is not completely blocked by TEA. Figure 3B presents plots of specific conductance (see figure legend) versus command voltage (Vc) for the same neuron (symbols). The data were fitted with a single Boltzmann function as given in the figure legend (Fig. 3B, lines).



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Fig. 3. La3+ effects on IA. A: superimposed responses of 1 neuron recorded in saline selective for IA to depolarizing steps from Vh -80 to +15 mV before and in the presence of La3+ at the concentrations indicated. La3+ reduces and slows the rise and decay of the transient peak. B: specific conductance (GA) is plotted against voltage (Vc) of the depolarizing step eliciting IA in control saline and saline with 1, 10, and 100 µM La (symbols, see legend in C; arrow indicates points from traces shown in A). GA was calculated as GA = [IA/ (Vc - EK)]/Cm, where EK, the calculated Nernst potential for K+ was -83 mV, and Cm, cell membrane capacitance, was obtained from the autocompensation of the EPC9, for this cell, 138 pF. Lines represent a fit to the data of a single Boltzmann function, g = GMax/{1 + exp[(V1/2 - Vc)/s]}. Note that maximum conductance is reduced with increasing [La3+] (see also Fig. 4A), the G-V curves are shifted to more depolarized Vc, and their voltage sensitivity is decreased. C: plot of normalized conductance vs. Vc, average ± SE, n = 8 (symbols). Lines represent a fit to the values of a Boltzmann function fitted to the averaged data. The values of V1/2 and s increase with increasing [La3+], as seen for specific conductance in B (see Fig. 4B).

The major effects of the addition of La3+ can be summarized as follows.

1) As seen for the neuron providing the data of Fig. 3B, there is a reduction in the maximum IA current and conductance that could be obtained with depolarizing voltage commands. A summary of all the observations is shown in Fig. 4A, which plots the averaged observations of the reduction of maximum conductance by La3+ from eight neurons examined. The reduction relative to control saline is nearly linear when plotted against the log [La3+] and amounts to ~30% at 100 µM [La3+].



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Fig. 4. Summary of La3+ effects on IA activation. A: reduction of maximum IA conductance by La3+. The ratio of the maximum conductance observed in the La3+ saline to that in control saline is plotted vs. [La3+] (log scale) (n = 8). B: depolarizing shift of Boltzmann parameters of IA activation by La3+. Values of Vc for eliciting half-maximal conductance and the slope obtained from fits to the averaged, normalized GA-V curves shown in Fig. 3C are plotted against [La3+] (log scale).

2) There is a shift of ~15 mV to more depolarized voltages (a right shift) of corresponding points of the IA-V and GA-V curves in 1 µM La3+ and a further shift of this magnitude with each 10-fold increase of [La3+], as seen in Figs. 3, B and C, and 4B and further described subsequently.

Changes in current amplitude might result from a change in relative permeability or driving force for permeant ions. Tail currents in response to repolarizing commands to a series of voltages after IA activation were therefore examined to test whether La3+ altered the reversal potentials. La3+ did not appreciably alter the reversal potential of nearly -80 mV, and thus it can be concluded that it does not alter the selectivity of the channel (data not shown). This value of the reversal potential is in good agreement with the calculated EK, thus supporting the identification of the current as a K+ current.

Cd2+ (1 mM) had no effect on IA, whereas Zn2+ (200 µM) and Pb2+ (100 µM) produced small (<10 mV) right shifts of the I-V curves. Removal of the 26 mM Mg2+ present in the normal saline had no effect (data not shown).

La3+ effects on IA activation

To further characterize the effect of La3+ in right shifting the voltage dependence of IA activation, the responses of different neurons tested in control and the [La3+] were normalized with respect to the maximum conductance observed for the particular neuron in each condition. The observations for the normalized GA-V curves averaged at each Vc (n = 7-8) are shown in Fig. 3C (symbols, with error bars, ±SE). Each of the normalized curves was fitted with a single Boltzmann function (Fig. 3C, lines). The values of the Boltzmann parameters used to fit the averaged normalized GA-V curves are plotted against log [La3+] in Fig. 4B. The V1/2, a measure of the right shift of the voltage dependence of activation, increased approximately linearly with the log [La3+] from a control value of approximately +4 mV to approximately +49 mV in 100 µM La3+, ~15 mV per 10-fold increase in La3+. Voltage sensitivity was reduced by the presence of La3+ as indicated by an increase in the slope factor, from 15 mV in control saline to 22 mV in 100 µM La3+.

La3+ effects on IA steady-state inactivation

The effect of La3+ on the voltage dependence of steady-state inactivation was examined with a double-pulse regime as illustrated in Fig. 5. A 300-ms hyperpolarizing or depolarizing prepulse (-100 to +20 mV) from Vh -70 mV was followed by a test depolarization chosen to evoke ~80% of the maximal IA, +20 mV for control saline, +30, +45, and + 60 mV for the 1, 10, and 100 µM La3+ salines. Previous studies (Meyers et al. 1992) have shown that 300 ms is ~10 times the maximum duration required to obtain the full extent of a change in steady-state inactivation, regardless of the voltage. A selection of the IA current traces from a typical experimental series is shown in Fig. 5A. In control saline, the responses after prepulses to -70 and -55 mV are superimposable while in saline with 10 µM La3+ responses after prepulses to -55 and -40 mV are superimposable. Figure 5B plots the fraction of the IA current in the absence of a prepulse over that after a prepulse versus the prepulse voltage. The prepulses from -100 up to -55 mV were without effect on IA under any conditions, inactivation reached its maximum (i.e., IA was minimal) with prepulses to -10 mV in control saline or 1 µM La3+-saline, and more depolarized prepulses were required with increasing [La3+]. There was consistently a residual outward current of ~20% that failed to inactivate. The curves were fitted by a single Boltzmann function, and the parameters are plotted in Fig. 5C. These indicate that, although for 1 µM La3+ the curves were not shifted, the voltage dependence of steady-state inactivation at 10 and 100 µM La3+ is right shifted by ~15 mV for each 10-fold increase in [La3+]. The slope is approximately doubled in 10 µM La3+ relative to the control value but not increased by a similar amount at 100 µM.



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Fig. 5. Effects of La3+ on steady state inactivation. A: example of records to evaluate effects of prepulse depolarization on IA. The voltage regime is shown; a 300-ms prepulse (incrementing values from -55 to -10 mV) is followed by a test pulse at Vc eliciting ~80% maximal IA (+20 mV for control, +30, +45, +60 mV for 1, 10, 100 µM La3+ salines). Five superimposed traces are shown in control saline (top) and in 10 µM La3+. B: fractional reduction of IA tested after a prepulse compared with IA in the absence of a prepulse is plotted vs. the prepulse voltage as recorded in control saline and 3 concentrations of La3+ (symbols, see legend). The data were fitted with Boltzmann functions (lines). C: Boltzmann parameters fitted to the data of B are plotted vs. [La3+] (log scale); the parameters show a depolarized shift for 10 and 100 µM La3+.

La3+ effects on IA kinetics

Inspection of the IA responses (e.g., Fig. 3A) showed that addition of La3+ not only reduced the amplitude but slowed activation as reflected by broadened peaks. The time to peak decreased with increasingly depolarized commands and reached a minimum, typically ~3 ms, for depolarizations to more than +45 mV in normal saline. In the analysis shown in Fig. 6A to compare different neurons the time to peak for each voltage command is normalized relative to the asymptotic minimum in control saline at +60 mV. The average (n = 4-8) for each Vc for all the neurons is plotted versus Vc. Saline with 1 µM La3+ showed little effect on the time to peak, whereas higher [La3+] right shifted the curves. The failure to show a right shift of the plot of relative time to peak versus Vc in 1 µM La3+ could reflect that GA is maximal and reduced only ~10% in 1 µM La3+ at this Vc. With sufficiently large depolarization, the same asymptotic minimum time to peak as in control saline was obtainable in the La3+ salines.



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Fig. 6. Effects of La3+ on kinetics of IA activation and fast inactivation. A: time to peak is severalfold slower in 10 or 100 µM [La3+] at Vc <+60 mV. Time to peak IA was normalized for each neuron to that in control saline at Vc +60 mV for each Vc. The averaged normalized values (n = 6) are plotted vs. Vc for control and 3 concentrations of La3+ (see legend). B: fast inactivation of IA is slowed by La3+. The time constants fitted to the initial decay from peak IA were normalized to that at Vc +90 mV in control saline for each neuron for each Vc in each saline and plotted as in A.

The rate of the fast, initial phase of inactivation that is characteristic of IA is also slowed by the presence of La3+. The voltage dependence of the rate of inactivation was evaluated by fitting a single exponential to the initial decline of IA responses to a series of commands. To compare observations from the several neurons, the values of the time constants at each Vc were normalized with respect to the asymptotic minimum observed for the neuron in control saline (+90 mV). The averages of these values (n = 4-8) are plotted versus Vc in Fig. 6B for the control saline and the three [La3+]. This analysis shows a right shift of the curves with increasing [La3+]. The Vc at which tau  is doubled is shifted ~20, 40, and 60 mV more positive for 1, 10, and 100 µM La3+ respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study has shown that La3+ at micromolar concentrations, in addition to its well-recognized ability to block membrane Ca2+ conductances, inhibits the rapidly inactivating K+ current (IA) of crab secretory neurons. The inhibition of IA is selective, as mM concentrations are needed to observe effects on the delayed rectifier K+ current. The [La3+] needed for half-maximal blocking of IA is similar to that for blocking the calcium current (ICa) of these neurons and is between 1 and 10 µM. La3+ is the most effective known blocker for the crab ICa, with Cd2+ having nearly equal potency (Richmond et al. 1995). La3+ produces a concentration- and voltage-dependent block of IA; the maximum conductance and current that can be elicited is reduced, and the current- and conductance-voltage relations are shifted to more depolarized values.

The plot of normalized time constants of the decay from peak IA versus Vc shows a depolarizing shift with increasing [La3+] that is also similar to that for the G-V relations (~15 mV/10 × increase in [La3+]). This is consonant with observations linking fast, N-type inactivation to activation (Hoshi et al. 1991; Zagotta and Aldrich 1990). Steady-state inactivation, as observed here with 300-ms prepulses, may also be attributed to N-type, as contrasted with C-type, inactivation. Thus a right shift of the fractional steady-state inactivation of IA versus prepulse voltage would be expected, as is observed. The lack of a change for the observations in 1 µM La3+ may be attributable to the choice of Vc for the test pulse, the more depolarized value used in the La3+ saline (+30 rather than +20) having compensated for the ~15 mV right shift of the G-V curve relative to control saline for 1 µM La3+.

The prepulse regimes for analysis of steady-state inactivation show a consistent 20% of the outward current that remains under all conditions. This corresponds to the current that is seen to remain at the end of long (>100 ms) depolarizations, whether IA is obtained by subtraction of records at different Vh or recorded after blocking IK with TEA (Fig. 1), as for the data considered in this paper. It is unclear whether this should be regarded as representing reactivation of conductance through IA channels or current through IK or other channels, a question that would require single-channel recording to resolve. Although this residual conductance was included as a parameter in fitting the Boltzmann parameters to data for steady-state inactivation, no other correction for this possible contamination of IA with other outward current was made in the analysis.

Our conceptual model of La3+ interaction with the IA channel visualizes repetitive binding and unbinding of a La3+ ion at a site part way into the pore channel. The right shift of voltage dependence as well as the voltage-dependent block of current is suggested to result from interactions with gating mechanisms when La3+ is bound at a specific site and obstruction of the pore by the La3+ when present in the channel. The ability of large depolarizing clamps to relieve block might result from the pulse expelling the blocking ion from the pore or the increased electrical driving force on K+ permitting it to dislodge La3+ as it moves outward through the pore.

Such a model is closely analogous to that developed by Woodhull (1973) for block of conductance in Na+ channels by protons. The assumptions made for development of that model appear reasonable for the case of La3+ block of the IA channel (for an application of this model to La3+ block of a barley-root voltage-dependent K+ current see Wegner et al. 1994). Figure 7 presents fits to a Woodhull model of the data used for Fig. 3. The fit to the observations are best for 1 µM La3+ and become poorer with increasing [La3+], especially for modest depolarizing commands. The Woodhull model provides estimates of the apparent affinity of the pore binding site in the absence of a voltage across the membrane and the distance across the electric field of the membrane of the binding site for different concentrations of the blocking ion. The affinities ranged from 1 to 8 µM for the 1- to 100-µM range of [La3+] examined, and distances varied from 0.2 to 0.34 of the membrane field (Fig. 7 legend).



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Fig. 7. Observed La3+ inhibition of IA compared with predictions of the Woodhull (1973) model. Data (symbols, see legend) for IA are from the experiment shown in Fig. 3; lines connect points calculated for each Vc and [La3+] with the equation Imodel = IcontrolKVc/([La3+] + KVc), where KVc = K0 mV exp(zdelta FVc/RT), and z (=3 for La3+), F, R, and T have their usual meaning. The parameters used for 1, 10, and 100 µM La3+ for the apparent binding constant for La3+ at Vm = 0 mV (K0 mV), estimated from the data, were 1.15, 3.1, and 7.8 µM, respectively; the distances of the binding site within the membrane voltage field, delta , were 0.20, 0.28, and 0.34.

In Woodhull's (1973) observations on [H+] effects on Na+ currents, right shift of the voltage dependence was separated from H+ block and was attributed to change of surface charge. However, an effect of La3+ on surface charge (Latorre et al. 1992) seems unlikely for the crab neurons; La3+ was effective at micromolar concentration in the presence of a high ionic strength saline and high concentrations of both Ca2+ (13 mM) and Mg2+ (26 mM). Rather we suggest that changes of the voltage dependence and voltage sensitivity of IA conductance result from an electrostatic interaction between gating charges and the ion when bound to a selective site near or in the pore. This explanation was proposed in a number of studies of effects of La3+ and/or of divalent cations on voltage-gated channels. These include observations in typical vertebrate salines (e.g., Spires and Begenisich 1994; Talukder and Harrison 1995) as well as of squid axons in high ionic-strength saline (Armstrong and Cota 1990; Gilly and Armstrong 1982a,b).

Although there appear to have been a number of studies comparing the effects of divalents on different types of K+ currents (see Mayer and Sugiyama 1988), relatively few examined the effects of lanthides. Ours is the first full report on crustacean neurons to our knowledge. In cultured hippocampal rat neurons (Talukder and Harrison 1995), La3+ differed from the other ions having effects at less than millimolar concentrations, Pb3+, Gd3+, Cd2+, and Zn2+, in its low threshold (~5 µM) and in producing a marked reduction of maximum IA current in addition to right shifting the activation and inactivation curves as did the divalents. La3+ and the others, unlike Zn2+, which caused a parallel right shift in the activation curves, decreased the voltage sensitivity of the activation curves. La3+ (and Pb3+), unlike Cd2+ and Zn2+, also inhibited the delayed rectifier current (IK), although at a much higher concentration (100 µM) in these hippocampal neurons. We report here that unlike in the hippocampal neurons Pb3+, Cd2+, and Zn2+ had little effect on the crustacean neurons.

Marked effects of La3+ on K+ currents but much less selectivity for IA was seen in a study of rat cerebellar granule neurons (Watkins and Mathie 1994). In the cerebellar neurons a separation of La3+ effects on activation and inactivation was observed, with inactivation voltage relations being right shifted more than activation so that with appropriate pulse regimes enhanced IA currents could be elicited. In both cultured rat superior cervical ganglion neurons and embryonic chick sympathetic neurons, 1 µM La3+ was seen to enhance transient outward current (Przywara et al. 1992). Although the I-V curves appeared to have been shifted to more polarized voltages, data on prepulse effects on steady-state inactivation were not presented. Thus the enhanced IA may represent the result of a depolarizing shift of the voltage dependence of steady-state inactivation.

Finding apparently different effects of divalents or lanthides on the voltage dependence of activation and fast inactivation raises the question of how independent these processes are. Values of the V1/2 and slope for activation and steady-state inactivation differ in all preparations examined, with the value of V1/2 usually more polarized and the slope factor smaller (voltage sensitivity greater) for inactivation. As briefly reviewed previously, although some of the di- and trivalent cations produce parallel shifts of activation or inactivation I-V curves, in several there are changes in the slope factor as well. The extent of the shifts and of changes in slope factor may differ for activation and steady-state inactivation, as seen in this study. In ours and the experiments discussed previously it was not feasible to analyze the effects of possible rapid time-course interaction between activation and inactivation. From analyses of macroscopic and single-channel currents of a cloned Drosophila shaker channel expressed in Xenopus oocytes, Zagotta and Aldrich (1990) concluded that inactivation occurs from a transition closed state penultimate to opening. This analysis thus links inactivation voltage relations and kinetics with those of activation. A more complex scheme involving 15 closed states rather than 5 in the path to opening was later used to model opening kinetics of a mutated shaker A1 K+ channel with a truncated N-terminus lacking fast (N-type) inactivation (Schoppa and Sigworth 1998; Zagotta et al. 1994). A similarly detailed model including inactivation has not yet been published to our knowledge. The existence of several genes coding transient K+ channels as well as a large number of splice variants of these gene products provide a structural basis for finding much variation in the biophysical characteristics of transient K+ channels and the influence on them of trivalent and divalent cations.

If La3+ blocks outward current at concentrations similar to those blocking ICa, its use to evaluate the presence of residual outward currents in studies of voltage-gated ICa is invalid. Our work suggests that it is important to also evaluate the effects of La3+ on outward currents in the absence of inward currents. It will be of interest to learn how general the selective inhibition of IA by La3+ may be.


    ACKNOWLEDGMENTS

We thank J. W. Labinia for preparing primary cultures of X-organ neurons as well as for unfailing technical assistance; Prof. Martin Rayner for critical comment on drafts of the mamnuscript; and Dr. Marc Rogers for helpful discussion.

This work was supported by the Cades Fund and by National Institute of Neurological Disorders and Stroke Grant NS-15453.

Present address of S. Duan: Neurology Service (VA-127), University of California, Veterans Administration Medical Center, 4150 Clement St., San Francisco, CA 94121.


    FOOTNOTES

Address for reprint requests: I. M. Cooke, Békésy Laboratory of Neurobiology, University of Hawaii, 1993 East-West Rd., Honolulu, HI 96822.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 23 October 1998; accepted in final form 29 December 1998.


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ABSTRACT
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