Central neurons have multiple types of voltage-dependent potassium channels, whose activation during action potentials shapes spike width and whose activation and inactivation at subthreshold voltages modulate firing frequency. We characterized the voltage-dependent potassium currents flowing during the action potentials of hippocampal CA3 pyramidal neurons and examined the susceptibility of the underlying channel types to inactivation at subthreshold voltages. Using acutely dissociated neurons that permitted rapid voltage clamp, action potentials recorded previously were used as the command voltage waveform, and individual components of potassium current were identified by pharmacological sensitivity. The overall voltage-dependent potassium current in the neurons could be split into three major components based on pharmacology and kinetics during step voltage pulses: I D (fast activating, slowly inactivating, and sensitive to 4-aminopyridine at 30 μm),I A (fast activating, fast inactivating, and sensitive to 4-aminopyridine at 3 mm), andI K (slowly activating, noninactivating, and sensitive to external TEA at 3–25 mm). The potassium current during the action potential was composed of approximately equal contributions of I D andI A, with a negligible contribution ofI K. I D andI A had nearly identical trajectories of activation and deactivation during the action potential. BothI A and I D showed steady-state inactivation at subthreshold voltages, but maximal inactivation at such voltages was incomplete for both currents. Because of the major contribution of both I Dand I A to spike repolarization, it is likely that modulation or partial inactivation at subthreshold voltages of either current can influence spike timing with minimal effect on spike width.
Although action potentials in squid axons are formed by just two types of voltage-dependent channels (Hodgkin and Huxley, 1952), vertebrate central neurons each express at least a dozen different types of voltage-dependent ion channels (Llinás, 1988; Brown et al., 1990; Hille, 2001). The expression in central neurons of multiple types of potassium channels in particular confers the ability to fire with a variety of patterns over a broad range of frequencies (Connor and Stevens, 1971a,b; Rudy, 1988;Storm, 1990; Hille, 2001; Rudy and McBain, 2001).
How do the many channel types present in a particular neuron work together to determine its firing properties? This issue has been addressed primarily by computer modeling using Hodgkin–Huxley-like equations, extended by the addition of many conductances, with equations for each conductance based on experimental analysis of voltage and time dependence (Connor and Stevens, 1971b; Huguenard and McCormick, 1992, 1994; Johnston and Wu, 1995; Locke and Nerbonne, 1997b). This approach has been powerful and informative, but it has limitations. For central neurons, the connection between experimental measurements and modeling is quite indirect. For example, most voltage-clamp studies of potassium channels in central neurons are based on measurements of kinetics using voltage steps far longer than a typical action potential (0.5–2 msec). Thus, modeling of events during the action potential is typically based on kinetic models whose behavior is based on extrapolations of kinetics that are actually measured on a far slower time scale.
Our goal was to directly measure the potassium current flowing during the action potential of hippocampal CA3 pyramidal neurons and to determine the relative contribution of different channel types to the overall current. We performed voltage-clamp experiments using experimentally recorded action potentials as the command waveform, a procedure that has been used previously in a variety of cell types to examine the flow of various currents during the action potential (Llinás et al., 1982; de Haas and Vogel, 1989; Doerr et al., 1989; Zaza et al., 1997; Raman and Bean, 1999). We then used pharmacology to distinguish various components of the overall potassium current. Our results fit well with previous studies in hippocampal pyramidal neurons (Storm, 1987, 1990; Wu and Barish, 1992, 1999) and other excitatory neurons (Locke and Nerbonne, 1997a,b; Kang et al., 2000), suggesting that the potassium currents known asI A andI D each contribute significantly to the repolarization of the action potential. In addition, we examined how the inactivation of I A andI D during slow subthreshold depolarizations affects their subsequent activation during action potentials.
MATERIALS AND METHODS
Cell preparation. Long–Evans rats (postnatal day 5–12) were anesthetized with isoflurane and decapitated, and brains were quickly removed and placed in ice-cold, oxygenated dissociation solution containing (in mm): 82 Na2SO4, 30 K2SO4, 5 MgCl2, 10 HEPES, 10 glucose, and 0.001% phenol red, buffered to pH 7.4 with NaOH. Hippocampi were dissected and cut with a McIlwain tissue chopper (Mickle Engineering, Gomshall, UK) into 350-μm-thick slices. The slices were transferred into the dissociation solution with 3 mg/ml protease (Sigma type XXIII; Sigma, St. Louis, MO), incubated at 37°C for 8 min, and then rinsed twice in dissociation solution with added 1 mg/ml trypsin inhibitor and 1 mg/ml bovine serum albumin (at 37°C). After enzyme treatment, the slices were stored in dissociation solution with trypsin inhibitor and bovine serum albumin at 22°C under a pure oxygen atmosphere. Slices were withdrawn as needed, and the CA3 region was dissected and triturated through a fire-polished Pasteur pipette to release single cells. Hippocampal pyramidal cells were identified morphologically by their large pyramidal shaped cell body (12–16 μm by 20–36 μm) with a thick stump of apical dendrite.
Recording pipettes. Electrodes were pulled from borosilicate glass micropipettes (VWR Scientific, West Chester, PA) with a Sachs-Flaming puller (Sutter Instruments, San Rafael, CA) to yield resistances between 1 and 1.5 MΩ. To reduce pipette capacitance (and facilitate optimal series resistance compensation), the shanks of the electrodes were wrapped with thin strips of Parafilm (American National Can, Greenwich, CT) to within several hundreds of microns of the tip.
Current-clamp recordings. Action potentials were recorded with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) in bridge mode. Resting membrane potentials typically ranged from −45 to −65 mV. We presume that the less-negative resting potentials may reflect depolarization caused by trauma during the isolation or caused by leak around the seal. To elicit full-blown action potentials, cells were hyperpolarized to potentials between −90 and −60 mV with steady injection of DC. Action potentials were evoked by short (generally 1 msec) current injections so that the period of current injection was over before the action potential. Voltage was filtered at 10 kHz (four pole Bessel filter), sampled at an interval of 10–25 μsec using a Digidata 1200A digital-to-analog (D/A) and analog-to-digital (A/D) converter and Clampex7 software (Axon Instruments), and stored on a computer hard disk.
Whole-cell voltage-clamp recordings. Currents were recorded with an Axopatch 200A amplifier (Axon Instruments), filtered with a corner frequency of 10 kHz (four pole Bessel filter), sampled at an interval of 10–50 μsec using a Digidata 1200A D/A and A/D converter and Clampex7 software (Axon Instruments), and stored on a computer hard disk. In some cases, currents were later digitally filtered with a corner frequency of 1 kHz (boxcar smoothing). Compensation (∼80%) for series resistance (typically ∼2.5 times higher than the pipette resistance) was routinely used. Seal resistances were typically 1–4 GΩ, and cells had input resistances between ∼100 and ∼900 MΩ after establishing the whole-cell configuration.
Solutions. The standard pipette solution for both current-clamp and voltage-clamp experiments was (in mm): 108 K2HPO4, 9 HEPES, 9 EGTA, and 4.5 MgCl2, buffered to pH 7.4 with KOH (Sodickson and Bean, 1996). To promote the stability of the recordings, 14 mm creatine phosphate (Tris salt), 4 mm Mg-ATP, and 0.3 mmTris-GTP were included in the pipette solution. Stocks (10×) of the creatine phosphate, ATP and GTP, were stored at −80°C. Standard external solution was Tyrode's solution containing (in mm): 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose, pH 7.4, with NaOH. Tetrodotoxin (TTX; Calbiochem, La Jolla, CA) was added at 0.3–1 μm to block sodium channels. To focus on currents through voltage-dependent potassium channels, we blocked calcium entry by replacing external Ca2+ with equimolar (2 mm) Co2+. We chose Co2+ replacement rather than blocking calcium entry with Cd2+ or La3+, because these both produce dramatic shifts in the voltage dependence of I A(Klee et al., 1995). However, even Co2+replacement probably produces a smaller shift of the voltage dependence of I A in the depolarizing direction compared with Ca2+ (Numann et al., 1987); thus, the degree of inactivation of I Aat subthreshold voltages may be somewhat underestimated.
After establishing the whole-cell configuration, cells were lifted from the bottom of the recording chamber, and extracellular solutions were delivered through an array of gravity-fed quartz capillaries (inner diameter, 145 μm) placed in front of the cell. Stock solutions of TEA (1 m), 4-aminopyridine (4-AP) (1 m), and TTX (0.3 mm) were prepared in deionized water and either stored at 4°C (TEA and 4-AP) or in aliquots at −20°C (TTX). All reagents, unless noted otherwise, were purchased from Sigma.
Leak subtraction. Correction for linear leak currents was done by subtracting a scaled current elicited by a 10 mV hyperpolarizing (or, at a holding potential of −110 mV, depolarizing) prepulse. For experiments using action potential waveforms as voltage commands, leak currents were defined by recording the currents in response to an inverted, scaled-down (by a factor of five) action potential used as command waveform, delivered from a holding potential of −80 or −90 mV.
Analysis. Data were analyzed and displayed using ClampFit6 (Axon Instruments), Microcal (Northampton, MA) Origin 5.0, and Igor Pro 3.12 (WaveMetrics, Lake Oswego, OR). All reported voltages are corrected for a liquid junction potential of −10 mV between the pipette solution and the Tyrode's solution (in which the pipette current is zeroed before sealing onto a cell), measured as described byNeher (1992). Statistics are given as mean ± SEM.
All experiments were performed at room temperature.
We began by recording action potentials in the dissociated neurons and examining the effects of various potassium channel blockers on action potential shape. Cells were held at resting potentials negative to −60 mV by injecting steady hyperpolarizing current [between 0 and 580 pA, with a mean of 128 (n = 66)], and single action potentials were elicited by 1 msec current injections (Fig.1). The average voltage threshold, measured as the least depolarized voltage (just after the current injection) for which an action potential fired, was −55 ± 1 mV (66 cells). Analysis of 255 action potentials from 17 cells yielded an average overshoot potential of +31 ± 2 mV, a maximal upstroke of 213 ± 18 mV/msec, a maximal downstroke of −64 ± 4 mV/msec, and a spike width of 1.4 ± 0.1 msec at 0 mV. These are similar to parameters reported for intact hippocampal pyramidal cells in brain slices recorded at room temperature (Bergles, 1995).
Figure 2 shows the effect of various potassium channel blockers on the action potential. Addition of 4-AP resulted in delayed repolarization of the action potential (Fig.2 A). With exposure to 30 μm4-AP, action potentials were 38 ± 10% (n = 7) wider (as measured by the area under the curve). At 2.5 mm, 4-AP had a more dramatic effect, increasing the action potential width by 310 ± 79% (n = 3). Exposure to 25 mm external TEA (Fig.2 B) also produced large effects, often leading to a sustained depolarization or plateau near −40 mV after the action potential. TEA had more pronounced effects on the later phases of repolarization than on the early phase (from the peak near +40 mV to ∼0 mV), whereas 2.5 mm 4-AP dramatically affected both phases. Interestingly, the effects on action potential shape of low 4-AP (modest broadening), high 4-AP (dramatic broadening, including early phase of repolarization), and TEA (dramatic broadening, but little effect on early phase) in CA3 neurons were very similar to those observed in callosal-projecting rat visual cortical neurons (Locke and Nerbonne, 1997b).
In adult hippocampal neurons studied in brain slices, blocking calcium entry or rapidly chelating intracellular calcium significantly slows the decay of the action potential, suggesting a prominent role for calcium-activated potassium current in action potential repolarization (Storm, 1987; Poolos and Johnston, 1999; Shao et al., 1999). In contrast, in the younger CA3 neurons we studied, removing external calcium (substituting cobalt) had relatively small effects on the action potentials (Fig. 2 C). Most commonly, the action potential became slightly wider in the initial phase of repolarization, consistent with a block of a small fraction of calcium-activated potassium current that contributes to initial repolarization, and slightly narrower in the later phase of repolarization, consistent with removal of a shoulder attributable to net inward calcium current. Possibly the role of calcium-activated potassium channels would be larger in the absence of the 9 mm EGTA present in the internal solution, although in adult cells, Storm (1987) found that EGTA was ineffective at disrupting repolarization, in contrast to the faster chelator BAPTA. In subsequent voltage-clamp experiments, we focused on purely voltage-activated potassium currents, using Tyrode's solution in which external Ca2+ was replaced by equimolar (2 mm) Co2+.
Voltage-dependent potassium currents elicited by step depolarizations
To characterize the components of voltage-dependent potassium currents sensitive to 4-AP and TEA, we began with experiments using voltage-clamp protocols using step depolarizations. Figure3 shows a voltage protocol used for one series of experiments, along with representative currents. Currents were elicited in Tyrode's solution with 1 μm TTX to block voltage-dependent sodium currents and with external calcium replaced by cobalt to block voltage-dependent calcium channels and calcium-activated potassium currents. Voltage steps from −90 mV to 0 mV activated outward current consisting of two kinetic components, an early transient peak, reached within a few milliseconds, and a maintained component with little decay for the remainder of a 200 msec test pulse. When a 50 msec prepulse to −40 mV preceded the test pulse to 0 mV, almost all of the initial transient current was removed, leaving a relatively slowly activating, slowly inactivating current (Connor and Stevens, 1971b; Numann et al., 1987; Klee et al., 1995). Subtraction of the current at 0 mV with and without prepulses yielded a current that activates quickly (time to peak of 5.2 ± 0.2 msec;n = 33 cells) and inactivates completely with a time constant of 15 ± 1 msec (n = 33 cells).
Using this protocol to distinguish between these two kinetic components of current, we found that they had different pharmacology. Figure4 A shows the dose–response relationship for inhibition of these components of current by 4-AP. Application of 4-AP at 30 μmhad practically no effect on the transient, prepulse-sensitive component of current, and half-block of this component required between 300 μm and 1 mm 4-AP. Both the kinetic characteristics of the current and the moderate sensitivity to block by 4-AP of the transient current are consistent with identification as A-type potassium current (I A). Effects of 4-AP on the maintained, nonprepulse-sensitive current were very different. At 30 μm, 4-AP reduced the sustained current, measured at the end of a 200 msec test pulse (“late current”) by 33 ± 3% (n = 19), and there was only modest additional block by concentrations ≤3 mm (which blocked by 48 ± 3%; n = 15). This high sensitivity to 4-AP of a slowly inactivating current is consistent with the potassium current now referred to asI D described previously in hippocampal pyramidal cells (Storm, 1988; Ficker and Heinemann, 1992; Wu and Barish, 1992; Bossu et al., 1996; Li and McArdle, 1997; Martina et al., 1998) as well as other neurons (McCormick, 1991; Surmeier et al., 1991;Locke and Nerbonne, 1997a; Martina et al., 1998). The additional block of late current at 3 mm 4-AP might reflect the presence of an additional sustained current component with low 4-AP sensitivity, which most likely represents the delayed rectifier potassium current I K (Storm, 1990).
External TEA at 1 mm had no effect on the transient, prepulse-sensitive current (Fig. 4 B). Increasing the concentration of TEA produced some inhibition, with block of 44 ± 2% at 25 mm TEA (n = 10) of this component of current. The concentration dependence of TEA sensitivity is consistent with approximately half of the transient current being sensitive to TEA inhibition, with half block of this component by 5 mm TEA. The late current was much more sensitive to TEA, with 1 mm TEA producing 39 ± 2% (n = 8) inhibition. Increasing the TEA concentration to 5 mm produced inhibition of 60 ± 5% (n = 6), and there was no additional effect of increasing the concentration to 25 mm (54 ± 6% inhibition; n = 10).
The experiment shown in Figure 5 examined in more detail the kinetics of the components of outward current (activated by a step from −90 to 0 mV) identified by the cumulative application of 30 μm 4-AP, 3 mm 4-AP, and 25 mm TEA. The component of current inhibited by 30 μm 4-AP shows rapid activation kinetics and slow inactivation (∼20–30% decay over 200 msec). Both these kinetic characteristics and the high sensitivity to 4-AP are consistent with the properties of the current named I Ddescribed in studies of hippocampal pyramidal cells in brain slices (Storm, 1990). The component of current identified by the further action of 3 mm 4-AP shows rapid activation and rapid inactivation. These characteristics, together with the sensitivity of this component to prepulse inactivation (Fig. 3) and its moderate sensitivity to 4-AP, are consistent with the current component named I A in hippocampal (Storm, 1990) and other neurons (Rogawski, 1985). The current sensitive to inhibition by 25 mm TEA (applied in the presence of 3 mm 4-AP) showed slow activation and no inactivation over 200 msec. These characteristics are consistent with the delayed rectifier current calledI K (Segal and Barker, 1984; Brown et al., 1990; Locke and Nerbonne, 1997). Our functional definition ofI K was based on applying TEA afterI A had been blocked by 3 mm 4-AP, because there was some TEA sensitivity of the inactivating, prepulse-sensitive current (which is probably primarily I A). There was a small component of outward current remaining with 3 mm4-AP and 25 mm TEA; we did not attempt to further characterize this current.
Together, these results are consistent with three distinct components of voltage-activated potassium current in isolated CA3 neurons. One current is rapidly inactivating and prepulse sensitive and is weakly sensitive to 4-AP. One is very sensitive to 4-AP, slowly inactivating, and not sensitive to a prepulse. A third is sensitive to external TEA, slowly inactivating, and not prepulse sensitive. These correspond well to the currents named I A,I D, andI K described previously in studies of hippocampal pyramidal cells in brain slices (Storm, 1990) and cultured neurons (Wu and Barish, 1992, 1999). These three major components of voltage-activated potassium current in CA3 neurons seem nearly identical to the components of potassium current distinguished in detailed studies on CA1 pyramidal neurons (Martina et al., 1998) as well as another central excitatory projection neuron, callosal-projecting visual cortical neurons (Locke and Nerbonne, 1997a).
Voltage-dependent potassium currents elicited by action potential waveforms
We subsequently examined currents elicited under voltage clamp using an action potential recorded previously as the command voltage. With unmodified Tyrode's solution, the ionic current elicited by the action potential waveform (Fig.6 A) consisted of a large inward current during the upstroke of the action potential followed by a large outward current during the repolarization phase of the action potential (Fig. 6 B). The relationship between the elicited current and the voltage trajectory during the action potential is seen by plotting the elicited current as a function of the voltage during the action potential (Fig. 6 C). The inward current was completely blocked by TTX, suggesting that the upstroke of the action potential is entirely caused by TTX-sensitive sodium current (although calcium channels were not blocked in this experiment). Application of TTX had no effect on the outward current after the peak of the action potential, consistent with inactivation of the sodium current being complete before the falling phase of the action potential. With the outward current isolated after block of sodium current, it can be seen that the activation of the outward current during the action potential is very abrupt. There is almost no current activated during the upstroke of the action potential until a voltage of approximately +30 mV is achieved (0.42 msec after the start of the action potential, counting from the time the voltage crosses the threshold of −55 mV). The outward current increases very rapidly during the time that the action potential is near its peak, increasing from ∼1 to 6 nA in 0.46 msec, during which time the voltage rises from +40 to +55 mV and back to + 40 mV. The outward current then declines smoothly during the falling phase of the action potential, reflecting both the decrease in driving force for potassium ions and also deactivation of potassium channels.
Experiments examining action potential-elicited currents require good voltage control of large currents with rapid kinetics. Evidence for good voltage control comes from the lack of difference in outward currents when the large inward sodium current is blocked. If there were series resistance errors or other loss of voltage control, the voltage seen by the cell would be artifactually depolarized during the flow of sodium current, resulting in larger potassium currents. Such effects were seen if higher resistance pipettes or inadequate series resistance compensation were used.
Sodium-activated potassium currents have been reported in some neurons (Dryer, 1994). The experiment in Figure 6 shows that such a current does not contribute significantly to repolarization of the action potential in CA3 neurons, because total potassium current was unchanged when sodium influx was blocked.
We used the pharmacological manipulations developed with step depolarizations to determine the contribution of various components of potassium current to the overall outward current elicited by the action potential waveform. An example is shown in Figure7. Application of 30 μm4-AP inhibited the peak outward current by 38 ± 2% (n = 22), and application of 3 mm4-AP inhibited the peak current by 84 ± 2% (n = 20). Adding 25 mm TEA (in the continuing presence of 4-AP) had relatively little effect, producing a detectable amount of additional block in 6 of 11 cells (∼2% of the peak control current). An alternative way to quantify the components of outward currents during the action potential is to examine the total charge carried by the various components of current, obtained by integrating the current over the course of the action potential waveform. Based on this analysis, the contributions to the overall charge byI D,I A, andI K were 39 ± 2% (n = 22), 49 ± 2% (n = 20), and 2 ± 1% (n = 11), respectively. A total of 11 ± 2% (n = 11) remained unblocked by the combination of 3 mm 4-AP and 25 mm TEA.
Figure 7 B plots I D andI A as a function of the voltage during the action potential. The pattern of flow ofI D andI A during the action potential waveform was very similar. As for total outward current during the action potential, both showed negligible activation before the peak of the action potential was reached (in this case, near +30 mV) and rapid activation immediately after the peak. The similar kinetics ofI D andI A during the action potential is consistent with the rapid activation kinetics of both during step depolarizations. In addition, bothI D andI A decayed at approximately the same rate during the falling phase of the action potential. Given the difference in inactivation kinetics of the two channels, this suggests that the decrease in the current can be attributed primarily to deactivation and, to a lesser extent, to a decrease in the driving force on potassium ions (approximately twofold greater at 0 mV than −45 mV).
How complete is activation of potassium channels during the action potential? Does activation reach completion before deactivation begins? We approached this question by testing whether maintaining the depolarization of an action potential produced larger currents (Fig.8). The activation ofI D andI A is particularly interesting in this context, so we isolated total I D andI A by block with 3 mm 4-AP. The voltage command consisted of a partial action potential, interrupted and extended at a voltage of +10 mV on the falling phase. This is the point on the action potential at which maximal outward current is normally reached. Extending the action potential resulted in a substantial increase inI D andI A. Peak current was reached after another ∼3 msec at +10 mV and was more than twice that reached on initially reaching +10 mV on the falling phase of the action potential. Thus, in aggregate, I A andI D are <50% fully activated during the action potential. In four experiments using this protocol, activation of I A andI D during the action potential was 46 ± 9% of that achieved with extended step to +10 mV.
The experiment in Figure 8 also suggests that there is little or no inactivation of I D andI A during a single action potential, because decay of the combined current is not detectable until the action potential has been extended for ∼3 msec at +10 mV, approximately three times its normal duration.
Activation of IA andID preceding a spike
Both I A andI D are believed to be activated at subthreshold potentials and to contribute to delayed firing of action potentials during slow approaches to threshold (Connor and Stevens, 1971a; Segal et al., 1984; Storm, 1988). Because of the slow inactivation of I D, this effect can last ≤10 sec (Storm, 1988). As in studies on more intact cells, we observed delayed firing when threshold was approached slowly in isolated CA3 neurons. Figure 9 shows an example in which action potentials were evoked by long (400 msec) injections of current. The delay of the first spike was determined for the least depolarizing current injection that caused action potential firing. In 42 experiments from 31 cells, the time to peak for the first spike ranged from 40 to 350 msec with a mean value of 49 msec. This is considerably longer than the average membrane time constant of 12 ± 11 msec (mean ± SD), suggesting that the delay reflects more than the time required to reach threshold. In 20% of the experiments, the time to peak for the first spike was >86 msec. This cutoff represents three times the mean membrane time constant plus 50 msec (approximately the time for I A to activate and inactivate). In the example in Figure 9, the membrane was charged to a subthreshold voltage of −58 mV after current injection. An ∼200 msec delay followed, during which the voltage first hyperpolarized and then depolarized until threshold was reached, and an action potential was fired. This sequence is consistent with a sequence of activation and inactivation of potassium current, presumably some combination of I A andI D, at subthreshold voltages.
The experiments like that shown in Figure 9 fit well with previous results in recordings from hippocampal pyramidal neurons in brain slices, suggesting that delayed firing of action potentials can result from activation and inactivation of I Aand I D at subthreshold voltages. How complete is this inactivation? This is an especially interesting question in light of the fact that I Aand I D are also the major currents contributing to the repolarization of the action potential. If inactivation were complete during slow approach to threshold, the repolarization of the resulting action potentials might be greatly altered. Therefore, we examined the time course of inactivation ofI A andI D at relevant voltages. We used voltage-clamp protocols in which the action potential command waveform was preceded by a prepulse of increasing duration. 4-AP and TEA were used to pharmacologically separate and quantitatively assessI A andI D. As shown in Figure10, there is substantial but incomplete suppression of I A andI D by rectangular prepulses to −45 mV (the threshold potential) of ≤3 sec duration. As expected from their kinetics, I A is affected more rapidly and to a greater extent than I D.
Some depolarizing voltage trajectories that were observed to occur preceding a spike do not resemble a rectangular voltage step but gradually lead up to threshold. Therefore, we also tested the extent of inactivation of I A andI D with a depolarizing ramp of increasing duration, up to a threshold voltage of −45 mV, and coupled to the action potential command waveform (Fig.11). When preceded by a depolarizing ramp to threshold, I A andI D flowing during a subsequent action potential were both reduced, with considerably more effect onI A thanI D. These data are summarized in Figure 12. Both prepulse protocols induced substantial inhibition of either current, with the square pulse inducing greater attenuation. In both cases, the time dependence was faster for I A than forI D, and inactivation of both potassium currents was incomplete. A steady state ofI A peak current amplitude was reached at 31% (square prepulse) and 45% (ramp) of control. Conversely, inactivation of I D saturated at 59% (square prepulse) and 75% (ramp) of control. These findings show that even sustained depolarizations to potentials close to threshold produce only partial inactivation of I D andI A, leaving a substantial fraction of these rapid rectifiers available for repolarization of the ensuing action potential.
Previous voltage-clamp studies using brain slices, cultured neurons, and acutely isolated neurons have identified three principal voltage-activated potassium currents in hippocampal pyramidal neurons:I A,I D, andI K (Gustafsson et al., 1982; Segal and Barker, 1984; Zbicz and Weight, 1985; Lancaster and Adams, 1986; Numann et al., 1987; Sah et al., 1988; Storm, 1988; Lancaster et al., 1991;Klee et al., 1995; Bossu et al., 1996). Our results fit well with these previous studies in finding that most of the voltage-activated potassium current can be assigned to these three components, each identified with a particular pharmacological and kinetic profile. The component identified as I D is highly sensitive to 4-AP, fast activating, and slowly inactivating.I A is weakly sensitive to 4-AP, fast activating, and fast inactivating. I Kis sensitive to external TEA, slowly activating, and virtually noninactivating.
Our results suggest that essentially all of the potassium current underlying repolarization of single action potentials in hippocampal CA3 neurons comes from I D andI A, with approximately equal contributions of each. The participation ofI D in action potential repolarization agrees with previous results showing effects of low concentrations of 4-AP on action potential width in hippocampal pyramidal neurons (Storm, 1988; Wu and Barish, 1992). Besides contributing nearly equal amounts of current during the action potential,I D andI A seem interchangeable (at least during the spike itself) in that they follow nearly identical trajectories of voltage- and time-dependent activation and deactivation during the action potential (Fig. 7). In both cases, the decline of current during the later phases of the action potential appears to be primarily attributable to deactivation (along with the decreased driving force) rather than inactivation, because there is very little inactivation of either current even if the action potential is artificially prolonged several-fold (Fig. 8). The lack of substantial inactivation of I A during a single spike is consistent with the observation of Keros and McBain (1997)that arachidonic acid speeds inactivation ofI A but does not affect spike width. It is also interesting that there is a significant surplus capability of potassium current from I D andI A during a single spike in that the combined current reaches only ∼40% of maximal activation (Fig. 8). However, our experiments were performed at room temperature, and it is possible that at physiological temperature, the degree of activation of both I D andI A during a spike might be higher. Both the participation of two different channel types and the reserve capability can be considered safety factors tending to ensure rapid repolarization.
It is possible to make working hypotheses for the molecular basis of the potassium channel subunits making up the various components of potassium current in hippocampal pyramidal neurons. Kv2.1 is a likely candidate for I K, because Murakoshi and Trimmer (1999) found that delayed rectifier current could be inhibited by intracellular application of antibodies to Kv2.1, and Du et al. (2000) showed that treatment of CA1 neurons with antisense oligonucleotides directed against Kv2.1 reduced a delayed rectifier similar to the I K component in dissociated neurons. The lack of effect on single spike width of antisense treatment (Du et al., 2000) fits well with our finding of negligible activation of I K during an action potential. In agreement with the pharmacology ofI K, Kv2.1 channels expressed heterologously in mammalian cells are blocked only weakly by 4-AP, with a half-blocking concentration of 3 mm (Shi et al., 1994). The kinetic and pharmacological properties ofI K as we recorded it in acutely isolated CA3 neurons match very well with those of a corresponding component of potassium current in nucleated patches from CA1 pyramidal neurons in brain slice (Martina et al., 1998) proposed to correspond to Kv2 channels. Because CA1 pyramidal neurons express Kv2.2 as well as Kv2.1 subunits (Martina et al., 1998), it is very possible that the channels underlying I K might be heteromultimers (Du et al., 2000).
Kv4 family subunits are likely candidates for channels contributing toI A. CA3 pyramidal neurons express both Kv4.2 and Kv4.3 subunits (Serodio and Rudy, 1998). In several types of central neurons that have been examined, the amplitude ofI A is correlated with the level of expression of mRNA for Kv4.2 subunits (Song et al., 1998; Tkatch et al., 2000), and in both sympathetic neurons (Malin and Nerbonne, 2000) and cerebellar granule neurons (Shibata et al., 2000),I A is eliminated or greatly reduced by transfection with dominant negative Kv4.3 subunits, expected to eliminate currents from all Kv4 family channels. In CA1 neurons, which, unlike CA3 neurons, do not express significant levels of Kv4.3 (Serodio and Rudy, 1998), both expression of Kv4.2 immunoreactive protein andI A are dramatically reduced in regions of heterotopia induced by prenatal injections of methylazoxymethanol, consistent with a major role for Kv4.2 subunits in the generation ofI A (Castro et al., 2001). As in a subset of sympathetic neurons (Malin and Nerbonne, 2001), a component of I A in CA3 neurons might also originate from Kv1 family subunits, which, together with β1 subunits, can produce an I A-like current (Rettig et al., 1994); this might account for the component ofI A blocked weakly by TEA.
The molecular identification of I D in hippocampal pyramidal neurons is less certain. This current has kinetic properties of fast activation and slow inactivation and is blocked by 30 μm 4-AP. These properties would fit with channels made by Kv3.1 subunits (Grissmer et al., 1994; Martina et al., 1998), but few hippocampal pyramidal neurons appear to express detectable levels of Kv3.1 subunits (Weiser et al., 1995; Martina et al., 1998; Du et al., 2000). Channels formed by Kv1.5 subunits are also candidates for I D in hippocampal pyramidal neurons. Kv1.5 polypeptides are expressed in the cell bodies of CA3 neurons (Maletic-Savatic et al., 1995) and can underlie rapidly activating, slowly inactivating currents with high sensitivity to 4-AP (London et al., 2001).
In addition to their role in action potential repolarization, it is believed that both I A andI D can undergo a sequence of partial activation and inactivation at subthreshold voltages and thus tend to produce a delay in spike firing (Connor and Stevens, 1971a; Segal et al., 1984; Storm, 1988; Luthi et al., 1996). Indeed, in our experiments on isolated neurons, such a delay of action potential firing was observed when just enough sustained current was injected to depolarize the membrane to threshold (Fig. 9). Although the roles ofI A andI D in spike repolarization appear to be interchangeable, this is unlikely to be true of their roles at subthreshold voltages, where differences in inactivation rates and completeness are expected to have more significant functional effects. At subthreshold voltages, we found that inactivation ofI A was both faster and more complete than that of I D. We did not attempt to resolve activation of I A andI D during the lead-up to action potentials. In principle, such currents might be small if channels can inactivate at subthreshold voltages without first activating. This is true for cloned Kv4.2 channels (Bähring et al., 2001), but this important issue has apparently not yet been explored for nativeI A andI D in central neurons. The nonmonotonic change in voltage during the lead up to the action potential with just-threshold current injections suggests that one or both currents undergo partial activation followed by inactivation, but the currents needed to produce the changes in subthreshold voltage are probably just a few picoamperes.
One consequence of having two different channel types contributing equally to repolarization is to minimize effects on repolarization rate when either current is reduced. This may be especially important because both I D andI A also play roles in subthreshold phenomena. Thus, one current can undergo a cycle of partial activation and inactivation leading up to the spike without resulting in elimination of the current available for repolarizing the spike. Because I A andI D contribute approximately equally to the current underlying repolarization, we can observe the consequences of reducing this current by half by examining the effects of 30 μm 4-AP, which blocksI D but notI A. This resulted in a relatively modest broadening of the action potential. In adult pyramidal neurons, calcium-activated potassium current through BK channels also contributes significantly to repolarization (Storm, 1987; Poolos and Johnston, 1999; Shao et al., 1999), which would further minimize the effects on spike repolarization of reducing eitherI D andI A alone.
Both I D andI A are known to be modulated by transmitters and second messenger systems (Nakajima et al., 1986;Deadwyler et al., 1995; Keros and McBain, 1997; Hoffman and Johnston, 1998; Colbert and Pan, 1999; Wu and Barish, 1999; Mu et al., 2000; Lien et al., 2002). The redundancy of their roles in spike repolarization means that modulation of either one may be able to produce significant functional effects at subthreshold voltages without compromising rapid spike repolarization.
This work was supported by National Institutes of Health Grants NS36855, NS38312, and HL35034 and by Fonds zur Förderung der wissenschaftlichen Forschung Grant J1853-MED. We thank Dr. Marco Martina for comments on this manuscript.
Correspondence should be addressed to Bruce P. Bean, Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115. E-mail:.