| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
The Center for Hearing Science,1 Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and 2Department of Otolaryngology/Head and Neck Surgery, The University of North Carolina, Chapel Hill, North Carolina 27599
 |
ABSTRACT |
|---|
|
10 mV positive to the second and has second-order kinetics. The second component activates with first-order kinetics. These two components also contribute to two kinetically distinct currents upon deactivation. The kinetic behavior of IHT was indistinguishable amongst cell types, suggesting the current is mediated by the same K+ channels amongst VCN neurons. Together these results provide a basis for more realistic modeling of VCN neurons, and provide clues regarding the molecular basis of the three K+ currents. |
|
INTRODUCTION |
|---|
|
): a rapidly inactivating A-type current (IA), a rapidly activating, slowly inactivating low-threshold current (ILT), and a non-inactivating high-threshold current (IHT). Although these currents have been described before (Manis and Marx 1991; Manis et al. 1996), their detailed kinetic behavior has not been elucidated. Because the kinetic behavior of K+ currents differs amongst the various known channel types (Chandy and Gutman 1994; Coetzee et al. 1999; Rudy 1988), kinetic analysis can provide insight into the identity of each participating channel type. In addition, kinetic analysis can provide critical information necessary to understand how and which channels regulate the subthreshold integration of synaptic inputs as well as the generation of complex action potential patterns. In this paper, we describe the kinetics of IA, ILT, and IHT in VCN neurons. We use these data to construct mathematical models of each current, which are compared directly to the experimental currents. The kinetic descriptions are used collectively in the following paper to simulate the electrical behavior of VCN neurons (Rothman and Manis 2003b). |
|
METHODS |
|---|
|
Procedures to isolate VCN neurons are as described in our previous paper (Rothman and Manis 2003a). For the data presented in this paper, all whole cell voltage-clamp recordings were made with an Axopatch 200 amplifier at 22°C. The extracellular solution [(in mM) 130 NaCl, 5 KCl, 2.5 CaCl2, 1.3 MgCl2, 30 glucose, and 10 HEPES] contained TTX and Cd2+ to block sodium, calcium, and calcium-dependent currents. Electrodes were filled with a K-gluconate solution [(in mM) 130 K-gluconate, 4 NaCl, 1 EGTA, 5 sucrose, 10 HEPES, and 4 Mg2ATP], having resistances in the range 3–10 M
. Only cells with stable 75–95% online compensation were included in the analyses. Under these conditions, the voltage-step rise time 
s was in the range 8–40 µs.
Kinetic analysis
Our kinetic description of a membrane current follows the classical formalism of Hodgkin and Huxley (1952), where the relationship between the channel conductance and measured current is defined as
 | (1) |
the order of activation, b the inactivation state variable, Vr the current's reversal potential, and V the membrane potential. The rate of change of states a and b are governed by the following first-order differential equation
 | (2) |
is the state time constant, and x
its steady-state value (i.e., the value of x when t >> x). Although this equation is different from the original HH formalism, in which x is expressed in terms of "opening" and "closing" rate constants 
and 
, it is nevertheless mathematically equivalent when x = /( + ) and x = 1/( + ). Because the solution to Eq. 2 for a voltage step at t = 0 is
 | (3) |
 | (4) |
and b by a Boltzmann function of the form
 | (5) |
= 0.5, and k is the slope factor that determines the steepness of the Boltzmann function. To simplify fitting the Boltzmann relationships to our current data points, we used a modified Boltzmann function in which conductance has been translated into current
 | (6) |
Time constants as a function of voltage were fit to a bell-shaped function of the form
 | (7) |
= C exp((V + 60)/V) and = C exp(-(V + 60)/V). Statistics
Statistical significance was assessed using a two-sided t-test for unpaired samples at the significant level P indicated. Significant differences are denoted with asterisks as follows: (0.01 < P <= 0.05, *) (0.001 < P <= 0.01, **) (P <= 0.001, ***). All results are reported as means ± SE. Error bars in the various figures also represent SE.
|
|
RESULTS |
|---|
|
In this section, we present a quantitative description of the rapidly inactivating A-type current, IA. As stated in our previous paper, IA was found only in a subpopulation of VCN Type I cells, the Type I-t cells (Rothman and Manis 2003a).
ISOLATION OF IA. Figure 1 shows the voltage-clamp protocol used to isolate IA. In this protocol, alternating preconditioning voltage steps (Vpp) were used to either remove inactivation of IA (Fig. 1A; Vpp = -110 mV) or inactivate IA (Fig. 1B; Vpp = -40 mV). Depolarizing voltage steps (Vcmd) applied after Vpp then evoked currents with and without IA, respectively. Point-by-point subtraction of alternating current traces yielded IA in isolation (Fig. 1C). Because the more positive Vpp often activated small amounts of IHT and/or ILT in our population of Type I-t cells (Fig. 1B, arrowhead), a 2- or 3-ms hyperpolarizing step to -110 mV (Vint) was added after every Vpp to deactivate most of IHT and/or ILT before Vcmd. The addition of Vint also generated identical capacitative electrode currents at the beginning of Vcmd, which could then be eliminated by subtraction (Fig. 1C). Although Vint removed 2% of IA, it did not affect its subsequent kinetic analyses.
|
ACTIVATION AND INACTIVATION OF IA. To estimate the time course of activation and inactivation of IA, isolated current traces of IA, as obtained in Fig. 1, were fit to the following equation
 | (8) |
)(V - Vr). This equation was derived from Eq. 4 assuming a0 = 0 and b0 = 1 (i.e., all A channels are closed and all inactivation is removed at the start of Vcmd) and b = 0 (true for Vcmd > -50 mV; see Fig. 3C). Examples of fits to IA are shown in Fig. 2A, with time represented on a log scale. Because a number of current traces showed both fast and slow inactivation components, the inactivation variable b was modified to include both a fast and slow time constant (bf and bs), in which the fraction attributed by each component was set by f. In our initial analysis, the order of activation , which measures the degree of sigmoidal activation, was allowed to vary from trace to trace; it was found, however, that a value near 4 usually produced the best fits. Hence, was set to 4 for the entire analysis. Results from nine Type I-t cells are shown in Fig. 2B, where each variable in Eq. 8 is plotted versus Vcmd. As this figure shows, IA is strongly voltage dependent with respect to both activation (Imax and a) and inactivation (bf). However, the slow inactivation time constant (bs), with values in the range 18–300 ms, is only sometimes voltage dependent. In all cases, the fast component of inactivation was significantly larger than the slow component (f > 0.7), and in 70% of the cells it was the only component.
|
|
To compute the steady-state activation of IA, modified Boltzmann functions (Eq. 6) were independently fit to the Imax-V relations in Fig. 2B and averaged together. Mean values for gmax, V0.5, and k were: 61.5 ± 7.2 nS, -30.8 ± 1.2 mV, and 6.1 ± 0.6 mV, respectively (n = 6).
Steady-state inactivation of IA was next investigated with the prepulse protocol in Fig. 3. This protocol is similar to the one in Fig. 1, except Vpp was varied while Vcmd was held constant. Again, Vint was used to deactivate any sustained outward current that might have activated during Vpp and to provide identical capacitative transients at the beginning of Vcmd. Although not apparent in this figure, sequence B, which consists of the same repeated voltage step, was interleaved with sequence A, in which Vpp was changed. The interleaved protocol was used to detect time-dependent rundown of IA. Subtraction of traces in B from those in A resulted in isolated IA in C. From C, an estimate of steady-state inactivation was obtained by computing peak current as a function of Vpp (inset) and fitting the resulting I-V relations to Eq. 6 (see legend for details). Repeating the same analysis for nine other Type I-t cells (inset, thin lines) yielded the following mean values for V0.5 and k: -66.1 ± 1.6 and -6.9 ± 0.4 mV.
Recovery from inactivation of IA was studied with the twin-pulse protocol shown in Fig. 4A, where two voltage steps to the same potential (VS1 and VS3) were separated by a hyperpolarizing voltage step (VS2) of variable length (TS2). As indicated by the current traces in Fig. 4A, recovery from inactivation was related to TS2 in an exponential-like manner. In Fig. 4B, current traces evoked by VS3 are plotted concurrently after subtraction of IHT (see legend for details). From these current traces, recovery functions were obtained by computing peak current as a function of TS2 (inset). Close inspection of these recovery functions shows a significant voltage-dependent delay in removal of inactivation: for VS2 = -100 mV, the delay is 3 ms; for VS2 = -70 mV, the delay is 8 ms. Furthermore, after the delay, the functions do not follow a single-exponential time course, but a bi-exponential one. Although the recovery functions could be satisfactorily fit to a sum of exponentials, a product of exponentials was adopted instead, because it could be readily modeled with a two-state function in the form bc (see Fig. 5B)
 | (9) |
b and c are plotted versus VS2 (squares and triangles; n = 8), in which case both time constants show strong voltage dependence.
|
|
NUMERICAL RECONSTRUCTION OF IA. From the above analysis, the following kinetic model of IA was developed
 | (10) |
A is the maximum conductance, which we leave as a free parameter because the magnitude of IA varied from cell to cell. VK is the reversal potential of IA. Because VK was not experimentally determined for IA, it was set it to -70 mV, the experimentally determined VK of ILT and IHT (see Figs. 7C and 10B). Parameter a4 is the activation variable whose steady-state value (a)4 is defined by Eq. 5 (V0.5 = -31 mV, k = 6 mV; from Fig. 2B). Parameters b and c are the fast and slow inactivation variables whose steady-state values, b and c, are defined such that their product equals the mean inactivation Boltzmann function of IA; specifically, b = c = y1/2, where y is defined by Eq. 5 (V0.5 = -66 mV, k = -7 mV; from Fig. 3). Time constants b and c were derived from the data in Fig. 4C and are shown as bold lines in that figure. b describes the fast component of removal of inactivation at V < -50 mV (squares), and the fast component of development of inactivation at V > -50 mV (circles). c describes the slow component of removal of inactivation at V < -50 mV (triangles). For V > -50 mV, c is set to a large saturating value of 100 ms because it showed no signs of diminishing in the real data. a was derived from the data in Fig. 2B. At V < -50 mV, the behavior of a is unknown because deactivation of IA was not investigated. We therefore assumed a behaved qualitatively similar to b at V < -50 mV, as it did at V > -50 mV (Fig. 2B). Hence, model a = bf/10 for all V.
|
|
Model current traces of IA are shown in Fig. 5A, computed with the same voltage protocol in Fig. 1A. For comparison, traces of model IA are plotted against those of experimental IA (inset). Because the model parameters are derived from the average of several Type I-t cells, the traces do not overlap exactly. However, the comparison shows that the time course of activation/inactivation of model IA is similar to that of experimental IA.
Despite the use of two inactivation variables b and c, inactivation of model IA proceeds along an apparent single-exponential time course as if f = 1. However, the absence of a measurable slow component of inactivation (c) is expected: for V > -50 mV, the effective inactivation time constant is bc/(b + c), which reduces to b when c >> b. Experimentally, inactivation of IA proceeded along a single-exponential time course in 70% of the cells, and when a bi-exponential time course was detected, the slow component was significantly smaller than the fast component (see Fig. 2B, parameter f).
The effects of parameter c are however apparent during recovery from inactivation as shown in Fig. 5B, where four different recovery functions of model IA are plotted concurrently. For comparison, traces of model IA (—) are plotted against those of experimental IA (– – –; from Fig. 4B). Again, because the model parameters are derived from the average of several Type I-t cells, the traces do not overlap exactly. However, the comparison shows that the model recovery functions display an onset delay (sigmoidal kinetics) similar to the experimental recovery functions. These results demonstrate that a simple two-state model of inactivation, bc, can adequately describe the delay observed in the recovery from inactivation.
Kinetic analysis of ILT
In this section we present a quantitative description of the rapidly activating, slowly inactivating low-threshold current, ILT. The analyses of ILT has been restricted to VCN Type II cells; these cells show large, unambiguous signs of ILT (Manis and Marx 1991; Rothman and Manis 2003a).
ILT OBEYS FOURTH-ORDER ACTIVATION KINETICS. To obtain an accurate estimate of the kinetics of ILT, it was first necessary to determine its order of activation, . This was accomplished with the voltage-clamp protocol in Fig. 6A, where a command step to -50 mV (Vcmd), which activates ILT but not IHT, followed a 100-ms prepulse (Vpp) to various potentials below -50 mV. The response of the Type II cell at the bottom of Fig. 6A shows that, when Vpp > -80 mV, ILT appears to activate with first-order kinetics. However, when Vpp < -80 mV, ILT shows sigmoidal activation kinetics, indicating ILT is mediated by a channel with multiple closed states.
|
To determine the value of , current traces in Fig. 6A were simultaneously fit to the following equation
 | (11) |
z > 150 ms; see results below), and the window of analysis comparatively brief (20 ms), the exponential decay of inactivation was ignored during the fit, in which case z was set to z0 for all time. During the fitting procedure, w and w were shared between traces because theoretically they should be the same for the same Vcmd. To determine if a single value of could account for the observed kinetic behavior of ILT, was also shared between traces. The two remaining parameters, Imax and w0, were allowed to vary from trace to trace to account for changes in the level of inactivation (z0) and the level of activation at the start of Vcmd (w0; Fig. 6A, inset). Fits thus obtained are shown in Fig. 6A (—). Clearly, current traces were satisfactorily fit with a single value of (4.1). Repeating the same analyses for 10 other Type II cells yielded the following estimate of : 4.2 ± 0.1 (n = 11). Hence, ILT obeys fourth-order activation kinetics similar to the n4-kinetics of the delayed-rectifier current first described by Hodgkin and Huxley (1952).
The finding that ILT obeys fourth-order kinetics differs from a previous finding that it obeys first-order kinetics (Manis and Marx 1991). The difference in findings is simply due to the use of prepulses in this study that deactivate ILT to various degrees before stepping to Vcmd. Previously the kinetics of ILT were analyzed from a holding potential near -60 mV, in which case ILT was 10% activated (w0 = 0.1), and therefore appeared to obey first-order kinetics when stepped positive (see Fig. 6A, 3rd trace from top).
DELAY OF ACTIVATION SHOWS EXPONENTIAL TIME COURSE. Results from the previous section demonstrate ILT shows a delay in activation when stepped from potentials below -80 mV, similar to the delay observed in other delayed-rectifier currents (Hodgkin and Huxley 1952; Schoppa and Sigworth 1998; Young and Moore 1981). Because it has previously been shown that this delaying effect develops with an exponential time course (Begenisich 1979), it was of interest to determine if this was also true for ILT.
Figure 6B shows the prepulse protocol used to study the evolution of delay of ILT. In this protocol, a command step to -50 mV was preceded by a prepulse to -110 mV of variable length T, resulting in the 10 current traces shown as noisy dashed line. The delay of ILT is pointed out (
) and should not to be confused with the removal of inactivation (
). For small T, the delay was minimal, and ILT activated with an exponential-like time course (top). For long T, the delay was greater, and ILT activated with a sigmoidal time course (bottom).
To quantify the time evolution of delay, current traces of ILT were simultaneously fit to Eq. 11, with parameters w, and w shared between traces, and = 4 (see legend for details). Fits to the data in Fig. 6B are shown as black lines, again demonstrating ILT is satisfactorily fit to Eq. 11 when = 4. In Fig. 6B1, the value of w at the end of the prepulse, w0, is plotted versus the prepulse length T (
). The solid line is a single-exponential fit to the data ( = 1.4 ms), demonstrating the time evolution of the delay follows an exponential time course. Repeating the same analysis for another Type II cell similarly yielded = 1.2 ms. Thus the delay in activation of ILT can be accounted for by a simple w4-kinetic model. In this model, the current amplitude and time course of delay is determined by the initial open probability of the channel (w0), which in turn is determined by both the level (Fig. 6A) and length (Fig. 6B) of the conditioning prepulse.
ACTIVATION KINETICS OF ILT. Activation and deactivation kinetics of ILT were computed by fitting Eq. 11 ( = 4) to whole cell current traces elicited by depolarizing and hyperpolarizing steps from -60 mV (Fig. 7A) or -70 mV. Again, by limiting the analysis to V < -40 mV, contributions from IHT were minimized. For a number of cells, a hyperpolarization-activated inward current, Ih, began to activate at V < -80 mV; in these cases, fits were restricted to the first 10–20 ms, thereby minimizing contamination from Ih (which has a very slow activation time course on the order of seconds). Results are plotted in Fig. 7B, where w is plotted versus Vcmd for 33 Type II cells (). Together, activation and deactivation w describe a bell-shaped curve that peaks near -60 mV at 6 ms. However, the curve is not exactly bell-shaped in that activation w shows a steeper voltage dependence than deactivation w (2-fold). The difference in steepness arises from the fact that activation is described by a (1 - w)4 kinetic process, whereas deactivation is described by a single-exponential kinetic process (see Eq. 11). The curved solid line in Fig. 7B is a fit to Eq. 7 (see legend for values). Because the kinetic analysis of ILT was limited to V < -40 mV, w was given a lower limiting value of 1.5 ms, corresponding to the lowest value at -40 mV. However, it very well may be that w falls below 1.5 ms for V > -40 mV.
INACTIVATION OF ILT. Of the 49 cells classified as Type II in our study of VCN neurons, 41 showed visual signs of slow inactivation in their outward current traces at V > -50 mV (for example, see Rothman and Manis 2003a; Fig. 2C). For the majority of these cells, development of inactivation was too slow to allow an accurate estimate of its time course during the 100-ms window of observation (z > 150 ms; n = 28). For the remaining cells, development of inactivation was fast enough to allow single-exponential fits to its time course; in these cases, z had values 30–150 ms from -50 to 0 mV, with largest values near -50 mV (n = 13; not shown).
Our finding that the activation level of ILT is sensitive to a preconditioning voltage step (Fig. 6, A and B; ) also indicates ILT undergoes inactivation. Indeed, 77% of the Type II cells investigated in this study showed sensitivity to a preconditioning voltage step. However, just as z showed significant variability, as described in the preceding text, the voltage dependence of inactivation of ILT (z) showed significant variability. In nine Type II cells, z behaved in a Boltzmann-like manner (Eq. 6) with a visible lower limit near 0.5 for V > -60 mV (Vh = -70.9 ± 2.2 mV, k = -10.0 ± 0.7 mV; data not shown). In 11 Type II cells, z behaved in a more shallow Boltzmann-like manner that only appeared to have a lower limit near 0.5 (Vh = -50.9 ± 3.6 mV, k = -15.7 ± 2.1 mV; data not shown). In 10 Type II cells, z behaved in a linear-like manner and therefore did not fit well to a Boltzmann function. In no instance was inactivation of ILT ever complete as it was for IA. What the variability in inactivation of ILT was due to is not clear.
For seven Type II cells, removal of inactivation was investigated with the twin-pulse protocol in Fig. 6B. Of these seven cells, four showed steady-state amplitude changes in response to a change in the interpulse time T. For the cell in Fig. 6B, removal of inactivation is denoted (). For small T, little inactivation was removed, and steady-state ILT (Imax) remained at low levels (bottom). For large T, a significant amount of inactivation was removed, and Imax rose to higher levels (top). In Fig. 6B2, Imax from Eq. 11 is plotted versus T. Here, changes in Imax reflect changes in the initial level of inactivation, z0, because gmax, V and Vr are constants. Clearly, Imax, and therefore z0, varies with an exponential time course. The solid line is a single-exponential function with z = 54 ms.
K+ SELECTIVITY OF ILT. Given Eq. 11, Vr of ILT could be estimated from the instantaneous I-V relationships in Fig. 7C where I = 0. Of the 33 Type II cells in this figure, 30 had sufficient data to compute Vr by linear interpolation (i.e., data points fell above and below the 0-current axis). Computed in this way, Vr was estimated at -69.9 ± 0.8 mV. The fact that this estimate is positive to the theoretical Vr (-80 mV, assuming perfect K+ selectivity) indicates either this estimate of Vr is imprecise, the ion channels that carry ILT are not perfectly selective for K+, or intracellular and/or extracellular K+ concentrations are not as predicted (local accumulations of K+, for example, could shift Vr positive). Similar observations of a more positive Vr have been noted elsewhere (Bal and Oertel 2001; Manis and Marx 1991; Rathouz and Trussell 1998). Nevertheless, theoretical and empirical Vr suggest ILT is predominantly carried by K+.
NUMERICAL RECONSTRUCTION OF ILT. From the preceding analyses, the following kinetic model of ILT was developed
 | (12) |
LT is the maximum conductance, which we leave as a free parameter because the magnitude of ILT tended to vary from cell to cell. Parameter VK is the reversal potential of ILT, set to -70 mV (Fig. 7C). Parameter w4 is the activation variable whose steady-state value (w)4 is described by the normalized Boltzmann function of the DTX-sensitive low-threshold current in Type II cells (Rothman and Manis, 2003a; Table 2) (V0.5 = -48 mV, k = 6 mV), and whose time constant, w, is described by the bell-shaped curve in Fig. 7B. Parameter z is the inactivation variable whose steady-state value z is described by the average of those inactivation functions that fit well to a Boltzmann function with steady-state offset (
) in the form
 | (13) |
= 0.5. The model inactivation time constant z is plotted in Fig. 8B (inset), and was derived from the following assumptions based on experimental observations: z is bell-shaped; z 50 ms at -110 mV (Fig. 6B2); z 100–300 ms at V > -50 mV; and z > 300 ms near rest.
|
Figure 8A shows model ILT first without inactivation ( = 1.0 in Eq. 13; LT = 170 nS). Here, the model shows the signature features of ILT (arrows) as described previously (Rothman and Manis, 2003a): a small, steady outward current at a -60 mV holding potential (1), a small deactivating inward current in response to voltage steps below VK (2), and rapid activation (5). Furthermore, model ILT shows sigmoidal activation kinetics (inset) comparable to that of the experimental data in Fig. 6A.
Figure 8B shows model ILT now with inactivation ( = 0.5; LT = 272 nS so that ILT at -60 mV is the same as that in Fig. 8A). The same features of ILT are visible (arrows 1, 2, and 5), but now slow inactivation is apparent in the current traces during Vcmd > -50 mV (arrow 3), as well as in the tail currents (arrow 5). Such signs of inactivation of ILT was observed in the majority of Type II cells.
Figure 8C shows model ILT ( = 0.5) when studied with the same twin-pulse protocol in Fig. 6B. The time constants for the time evolution of the delay are d = 1.1 ms at -110 mV and for the removal of inactivation are z = 53 ms at -110 mV. Again, the model accurately replicates the experimental data.
Kinetic analysis of IHT
In this section, we present a quantitative description of the non-inactivating high-threshold current IHT. As stated in our previous paper (Rothman and Manis 2003a), all VCN cell types possess IHT; however, only the Type I-c cells appear to have IHT as its sole outward current at V > -80 mV. Hence the analysis of IHT in Type I-c cells is considered the prototype. Direct comparisons are then made to the DTX-insensitive current (IDTX-) in Type I and Type II cells, and to IHT in Type I-t cells. These comparisons show that IHT is kinetically indistinguishable amongst VCN neurons.
ACTIVATION OF IHT. The time course of activation of IHT was slightly more complex than expected for a single conductance. We found IHT was better described by the sum of two components rather than a single component. This is demonstrated in Fig. 9A, where IHT is better fit to a two-component model (n2 + p) than to a one-component model (n2). Similarly for deactivation, IHT was better fit to the sum of two exponentials rather than a single exponential (Fig. 10, A and B). These results agree with the previous finding of two components in the tail currents of IHT (Manis and Marx 1991).
|
After investigating several two-component models of IHT, we found a n2 + p model was better than a n + p model, and no worse than a n3 + p model (Fig. 9B). Although it is true a product of variables, such as np, can adequately describe the activation of IHT (not shown), such a model was inadequate in describing the deactivation of IHT because the tail currents of a np model decay with a single-exponential time constant = np/(p + n), which reduces to n/ when p >> n. Another description might be a n + np model, used previously to describe the activation of KCNC1 currents in NIH-3T3 cells (Kanemasa et al. 1995), and activation of a high-threshold DTX-insensitive current in principal cells of the MNTB (Wang, LY et al. 1998). However, deactivating tail currents in this model also decay as a single exponential ( n/ when p >> n).
According to Eq. 4, activation and deactivation of a n + p model is described by the following equation
 | (14) |
 | (15) |
)(V - Vn) and Ip = gpp(V - Vp). On the other hand, when activated currents are deactivated by stepping to V < -45 mV, such as those in Fig. 10A, n and p can be assumed to be zero, and Eq. 14 reduces to
 | (16) |
(V - Vn) and Ip = gpp0(V - Vp). Results of fitting Eq. 15 ( = 2) to the activation of IHT in Fig. 9A are shown in Fig. 9C. As Fig. 9C shows, n < p for all V and In > Ip for V > -20 mV. Furthermore, In appears to activate positive to Ip. This is apparent when V0.5 values of In and Ip are compared, computed from Eq. 6: -13 versus -26 mV, respectively. Fitting Eq. 16 ( = 2) to the deactivation of IHT in Fig. 10, A and B, gave similar results: n < p; In > Ip; and In activates positive to Ip. A summary of activation and deactivation analyses for a total of 16 Type I-c cells is shown in Fig. 11, where n is plotted in A, p in B, and n2 and p in C. For V > -45 mV, data are from activation analysis (Fig. 9), and for V < -45 mV, data are from deactivation analysis (Fig. 10). The n2 and p data in C were computed from In and Ip values, according to Eqs. 15 and 16, where n denotes n or n0, and p denotes p or p0. The bold lines in A and B are fits to Eq. 7, and the bold lines in C are average Boltzmann functions computed from the activation analysis (—) and the deactivation analysis (– – –). As A and B show, both n and p are described by bell-shaped curves that peak near -50 mV (peak values 4 and 17 ms, respectively). For all V, mean values of p are 5 times greater those of n. As C shows, n2 activates 10 mV positive to p for both activation and deactivation (V0.5 -15 and -25 mV, respectively). Hence, time constants and activation functions of the fast and slow component of IHT are well separated. These results are similar to those of Manis and Marx (1991) who reported p = 5.7n at -60 mV, and V0.5 = -13 and -24 mV for n and p, respectively (their n is equivalent to the n2 reported here; however, their n was multiplied by 2 to account for the difference in ).
|
IHT IS THE SAME AMONGST ALL VCN CELL TYPES. Using the same n2 + p kinetic model to analyze the dendrotoxin-insensitive high-threshold current (IDTX-) described in our previous paper (Rothman and Manis 2003a), we found few statistical differences between IDTX- and IHT in Fig. 11 (IDTX- data from 9 Type II cells and 4 Type I-i cells). Only for a few values of n near -55 mV were there statistical differences with P < 0.01 (data not shown). Hence, this comparison supports the conclusion that IHT and IDTX- constitute the same current.
Finally, we compared the n2 + p deactivation analysis of IHT in Type I-c cells to that of IHT in Type I-t cells. Although Type I-t cells possess both IA and IHT, deactivating tail currents of IHT in isolation could be obtained by stepping the membrane to V > -45 mV for 100–150 ms, at which time most of IA is inactivated (see Fig. 1C) and then stepping to V < -45 mV. Results of this comparison shows that deactivation of IHT in Type I-c and Type I-t cells is kinetically indistinguishable (data not shown) and therefore probably constitute the same current.
Although it was possible to obtain activating currents of IHT in isolation in Type I-t cells with a prepulse protocol (Vpp > -45 mV), the resulting current traces had to be fit to Eq. 14 rather than Eq. 15; in this case, there were too many free parameters to obtain a unique solution. Whether activation of IHT in Type I-t cells is best described by a n2 + p model is therefore not known. However, visual inspection of IHT in Type I-t cells shows clear signs of both a fast and slow component upon activation, indicating two components would be necessary to describe its time course.
K+ SELECTIVITY OF IHT. The reversal potential of IHT was estimated from tail currents evoked by hyperpolarizing steps from potentials above -45 mV (Fig. 10B). Because IHT constituted a fast and slow component, denoted In and Ip, two reversal potentials were computed: Vn and Vp. Specifically, Vn was estimated at the point where In = 0, and Vp where Ip = 0 (Eq. 16; Fig. 10B1). Computed in this way, Vn and Vp were estimated at -68.1 ± 2.1 and -67.4 ± 1.1 mV, respectively (n = 5 Type I-c cells). The same analysis of IHT in Type I-t cells resulted in similar estimates: -68.3 ± 1.0 and -63.6 ± 2.1 mV (n = 14 Type I-t cells). As did the same analysis for IDTX-: -70.6 ± 1.6 and -68.8 ± 3.8 mV (n = 5 Type II cells and 4 Type I-i cells; extracellular solution contained 10 or 100 nM DTX). Hence, both Vn and Vp are estimated near -70 mV, suggesting In and Ip are carried by K+.
Interestingly, while the above estimates of Vn and Vp are consistently near -70 mV for each cell type, they are not exactly the same. The small differences in average values are due to small differences in Vn and Vp on a cell-by-cell basis. This was sometimes apparent in the tail currents of IHT, in which case the fast component reversed sign before the slow component, or visa versa (not shown). This also suggests In and Ip are independent K+ currents.
NUMERICAL RECONSTRUCTION OF IHT. From the preceding analyses, the following kinetic model of IHT was developed
 | (17) |
HT is the maximum conductance, which we leave as a free parameter since the magnitude of IHT tended to vary from cell to cell. Parameter VK is the reversal potential of IHT, set to -70 mV. Parameter n2 is the fast activation variable with steady-state value n2 described by the Boltzmann function in Fig. 11C, and time constant n described by the bell-shaped curve in Fig. 11A. Parameter p is the slow activation variable with steady-state value p described by the Boltzmann function in Fig. 11C and time constant p described by the bell-shaped curve in Fig. 11B. The fractional amplitude factor 
is set to 0.85, a value derived from the activation and deactivation analyses described in the preceding text.
Model current traces of IHT are shown in Fig. 12A (HT = 150 nS). The voltage-clamp protocol is similar to that in Figs. 9A and 10A and therefore can be directly compared to experimental IHT. Fig. 12, B and C, shows the model's fast (In) and slow (Ip) components in isolation. Hence, a simple comparison between model IHT and experimental IHT clearly shows the sum of In and Ip describes IHT better than In in isolation. A model in which Ip is simply scaled would also provide a poor description of IHT because the tail currents would decay at a much slower rate than the experimental tail currents of Type I and II cells.
|
|
|
DISCUSSION |
|---|
|
In many ways, IA in VCN Type I-t cells fits the classic description of other "A" currents found throughout the central nervous system, including an activation threshold slightly below that of IHT, sigmoidal activation kinetics, exponential-like inactivation, complete inactivation at potentials above -50 mV, and a sensitivity to 4-AP (IC50 1 mM) (Manis et al. 1996; Rudy 1988). One notable difference, however, is in the kinetics of inactivation: whereas removal of inactivation of IA has usually been reported to follow a single exponential time course (Alekseev and Zaykin 1993; Bardoni and Belluzzi 1993; Belluzzi et al. 1985; Hsiao and Chandler 1995; Nagatomo et al. 1995), IA in this study followed a multiple exponential time course, similar to its sigmoidal activation kinetics. Only a few other studies have reported similar findings (Bilbaut et al. 1996; Rizzo and Nonner 1992). Whether the difference in inactivation indicates distinct "A" currents is not known. It may be the multiple-exponential time course has been overlooked in other studies. It is interesting to note a number of studies have reported a multiple-exponential time course in removal of inactivation of Na+ currents (Bezanilla and Armstrong 1977; Parri and Crunelli 1998; Sah et al. 1988; Sarkar et al. 1995). The simplest physical interpretation of these results is that IA has more than one sequential inactivation state; in which case, it must traverse two or more states before it can recover from inactivation. Hence the delay in removal of inactivation would be analogous to the delay in activation, where a channel must traverse more than one closed state before it can open.
Recently, a fast transient current (IKIF) has been described in pyramidal cells of the dorsal cochlear nucleus (DCN) (Kanold and Manis 1999). Although IKIF shows similar activation kinetics to IA in this study ( 1–5 ms, = 4), the two currents are considerably different in that IKIF shows a higher half activation and shallower voltage dependence (V0.5 -7 mV and k 17 mV) in comparison to IA (V0.5 -31 mV and k 6 mV), and the steady-state half inactivation of IKIF is 20 mV lower than that of IA. These differences suggest not only are IKIF and IA two distinct currents, but they serve two different roles in shaping the discharge pattern of CN neurons. Indeed, pyramidal cells in the DCN show very different discharge patterns compared to VCN Type I neurons (Manis 1990; Manis and Marx 1991; Oertel 1983).
Molecular identity of IA
To date, six K+ channel subunits exhibit rapid inactivation when expressed as homomers in Xenopus oocytes (KCNA4, KCNC3, KCNC4, KCND1, KCND2, and KCND3) and are therefore candidates for the molecular determinant of IA. KCNC4, however, shows only weak expression in the VCN (Fitzakerley et al. 2000; Weiser et al. 1994) and is therefore an unlikely candidate. KCNA4 and KCNC3 show strong expression in the VCN, as do KCND2 and KCND3 (Fitzakerley et al. 2000; Heck et al. 1997), and are therefore likely candidates. Our data suggest IA is kinetically distinct from IKIF and that IKIF is most likely mediated by KCND2 (Kanold and Manis 1999). However, further speculation about the molecular determinant of IA is difficult, given that K+ subunits exhibit significant variation in their kinetics when expressed with cofactors (An et al. 2000; Serodio et al. 1996) or when expressed as heteromultimers with other K+ channel subunits (Weiser et al. 1994).
ILT shows sigmoidal activation kinetics and slow inactivation
In this study, the use of prepulse protocols revealed two distinguishing characteristics of ILT in Type II neurons not previously reported: sigmoidal activation kinetics and slow inactivation. The finding that ILT undergoes sigmoidal activation suggests ILT is to be included in the class of K+ channels generally referred to as delayed rectifiers. However, because the delay is only observed at potentials below -80 mV, it probably has little functional relevance in an operating Type II cell. On the other hand, the absence of delay at potentials above -80 mV may be functionally important in that ILT activates almost instantaneously at these potentials, thereby influencing the membrane potential more directly than if there was a delay in activation.
The other distinguishing characteristic of ILT revealed by the prepulse protocols is its inactivation. Inactivation of ILT was distinct from inactivation of IA in that it had a significantly slower time course in both its development and removal and appeared always to be incomplete (i.e., ILT did not appear to decay to 0 steady-state levels as did IA). It is not clear whether the inactivation of ILT is functionally significant. If, for example, the membrane of a Type II cell is hyperpolarized to potentials below rest (via inhibitory inputs), inactivation of ILT will be partially removed. A depolarizing input thereafter will elicit larger ILT than when depolarized from rest, thereby altering the cell's response to excitatory inputs. On the other hand, given that a fairly large hyperpolarization is necessary to produce a dramatic change in inactivation (>20 mV for a 2% change in steady-state activation), perhaps larger than any real inhibitory input could manage; and given the low input resistance of Type II cells, it is unclear whether the inactivation could be substantially removed under physiological conditions. Modeling results, in fact, suggest no significant difference between simulations with or without inactivation of ILT, except for a small change in the resting membrane potential (not shown). Thus it appears that inactivation of ILT may have little functional significance.
Molecular identity of ILT
There are several lines of evidence pointing to the involvement of specific K+ channel subunits in the formation of ILT channels. First, very low (nanomolar) concentrations of DTX have been shown to specifically block KCNA1, KCNA2, and KCNA6 channels (Grissmer et al. 1994; Owen et al. 1997; Robertson et al. 1996). While DTX has not been tested against all K+ channel subunits, binding studies suggest it has a high affinity only for a few proteins with similar properties. Because ILT is also blocked by nanomolar concentrations of DTX [results from this study; also see Brew and Forsythe 1995; Rathouz and Trussell 1998], it is likely that KCNA1, KCNA2, and/or KCNA6 are part of the channel complex that gives rise to ILT. Second, both KCNA1 and KCNA2 have been shown by in situ hybridization to be expressed at high levels in VCN neurons (Grigg et al. 2000; Kues and Wunder 1992; VermaKurvari et al. 1997) and particularly in bushy cells (Fonseca et al. 1998). Immunocytochemical studies further indicate KCNA1 and KCNA2 proteins are present on neurons in the VCN (Wang et al. 1994). Third, in expression studies, KCNA1 and KCNA2 subunits show activation at more negative voltages than many other Kv family channels. However, the half-activation voltage reported in homomeric expression systems is still 20 mV positive to the measured half-activation of ILT in this study. Fourth, both KCNA1 and KCNA2 exhibit incomplete inactivation (Hopkins et al. 1994) similar to that seen in ILT. Finally, these subunits have been shown to be physically associated in native channels (Koschak et al. 1998; Wang et al. 1993).
Although it could be argued that ILT is mediated by channel types from the erg or the KCNQ families, the behavior of these channels is not well matched with ILT. erg channels, for example, activate and deactivate very slowly, reopen when repolarized from a depolarized potential and have a non-monotonic steady-state current-voltage relationship (Sanguinetti et al. 1996; Shi et al. 1997; Trudeau et al. 1999). Of the KCNQ family, only KCNQ2 and KCNQ3 are significantly expressed in brain. These channels have recently been suggested to correspond to the "M" current (Selyanko et al. 1999; Wang, HS, et al. 1998). Although it was originally suggested that ILT was similar to an M current based on its voltage dependence and kinetics (Manis and Marx 1991), it is clear that the kinetics of homomerically expressed KCNQ channels are quite different, including non-monotonic steady-state activation functions. Furthermore, homomeric KCNQ channels are insensitive to 4-AP (Yang et al. 1998), whereas ILT in bushy cells and their avian homologs can be blocked by 4-AP (Manis and Marx 1991; Rathouz and Trussell 1998; Reyes et al. 1994; Rothman and Manis 2003a; Schwarz and Puil 1997; Zhang and Trussell 1994). Finally, native M currents show first-order activation kinetics (Adams et al. 1982), whereas native ILT currents show fourth-order activation kinetics.
Because the bulk of the evidence supports the idea that ILT is most similar to KCNA1 and KCNA2, our working hypothesis is that other factors may be responsible for the differences in the voltage dependence of native ILT and KCNA1 or KCNA2 conductances. These include different basal phosphorylation states (Jonas and Kaczmarek 1996), glycosylation (Thornhill et al. 1996), redox states (Ruppersberg et al. 1991), or presence of distinct or other accessory subunits (Heinemann et al. 1994, 1996; McCormack et al. 1995; Nakahira et al. 1996; Rettig et al. 1994; Rhodes et al. 1997; Shi et al. 1996). The voltage dependence of native channels might be affected by association with subunits, glycosylation, or phosphorylation. Alternatively, it could depend on cooperative behavior of specific subunits (Smith-Maxwell et al. 1998).
Two components of IHT
Kinetic analysis of IHT in the Type I-c cells revealed two components of activation, a fast and a slow one, best described by the sum of two variables. The simplest interpretation of this result is that IHT constitutes two independent currents. Although there is no evidence to prove this with the given data, there are two lines of reasoning to suggest this is the case. First, the two components show significantly different voltage dependencies: in almost every Type I-c cell investigated, the slow component activated negative to the fast component and reached half activation (-25 mV) well below half activation of the fast component (-16 mV). Second, while the reversal potentials of the fast and slow component were similar, they were not exactly the same; this was sometimes apparent in the tail currents of IHT, in which case the fast component reversed sign before the slow component or visa versa.
The functional significance of two kinetically distinct high-threshold K+ conductances is not readily apparent. Manis and Marx (1991) suggest the slow component of IHT contributes to the length of the afterhyperpolarization following a spike, thereby affecting the interspike interval timing. This hypothesis was tested with the VCN model described in our next paper (Rothman and Manis 2003b) by simply removing the slow component from IHT (Eq. 17; = 1) and noting the change in interspike interval timing. Results of this analysis show no apparent change in either interspike interval timing or the shape of the action potentials. Hence these modeling results suggest the slow component of IHT may contribute little to shaping the discharge pattern of VCN neurons due to its small magnitude and/or slow kinetics.
VCN neurons probably share the same DTX-insensitive IHT
Comparison of IHT between Type I-c and Type I-t cells showed several similarities, including the presence of a fast and slow component with significant separation of half activation voltages. The time constants and activation curves of the fast and slow components showed few statistical differences between cell types. Hence these results suggest Type I-c and Type I-t cells express the same high-threshold conductance(s). One notable difference, however, was that IHT in Type I-c cells is approximately twice as large as it is in Type I-t cells (Rothman and Manis 2003a). This finding suggests Type I-t cells express fewer of the channels responsible for IHT. The functional significance of this finding is unknown. Our modeling results (Rothman and Manis 2003b) indicate a reduction in IHT from 150 to 80 nS (average values in the Type I-c and Type I-t cells, respectively) produces only a modest increase in action potential width along with a modest decrease in the threshold of action potential generation. It may be that IA in the Type I-t cells has usurped part of the role of IHT, in which case Type I-t cells can then down regulate their expression of IHT.
Comparison of IHT and the DTX-insensitive current in Type II and Type I-i cells also showed several similarities, including the presence of a fast and slow component with significant separation of half activation voltages. Again, the time constants and activation curves of the fast and slow components showed few statistical differences between the two currents. These results, along with those mentioned in the preceding text, have two implications. First, they imply IHT is DTX-insensitive (at concentrations of 
100 nM). Second, they imply all VCN cells express a kinetically similar DTX-insensitive high-threshold conductance.
Molecular identity of IHT
A good candidate for the molecular determinant of IHT is KCNC1. In the VCN, KCNC1 mRNA is expressed in most principal cells [except possibly octopus cells (Grigg et al. 2000)], including bushy and stellate cells (Perney and Kaczmarek 1997). When expressed in a mammalian cell line (NIH 3T3 fibroblasts), KCNC1 currents exhibit a fast and slow activation component (Kanemasa et al. 1995), similar in appearance to IHT. Furthermore, KCNC1 currents exhibit similar pharmacology to IHT in that they are insensitive to DTX but sensitive to 4-AP and TEA (Grissmer et al. 1994). However, there is one significant difference between the two currents: KCNC1 currents show a higher activation threshold and shallower voltage dependence (V0.5 35 mV, k 21 mV) in comparison to IHT (V0.5 -15 mV, k 7 mV). As pointed out by Perney and Kaczmarek (1997), the high activation threshold and shallow voltage dependence of the exogenous KCNC1 current could be explained by a post-translational modification of the protein that occurs in the native cells that does not occur in the fibroblast cell line. It could also be explained by a co-assembly of KCNC1 subunits with other K+ channel subunits.
|
|
ACKNOWLEDGMENTS |
|---|
|
Address for reprint requests: P. B. Manis, Dept. Otolaryngology/Head and Neck Surgery, 1123 Bioinformatics Bldg., CB#7070, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7070 (E-mail: pmanis{at}med.unc.edu).
|
|
REFERENCES |
|---|
|
Alekseev SI and Zaykin AV. Kinetic study of A-type current inactivation in Lymnaea neurons. Biochim Biophys Acta 1148: 97–107, 1993.[Medline]
An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, and Rhodes KJ. Modulation of A-type potassium channels by a family of calcium sensors. Nature 403: 553–556, 2000.[Medline]
Bal R and Oertel D. Potassium currents in octopus cells of the mammalian cochlear nucleus. J Neurophysiol 86: 2299–2311, 2001.
Bardoni R and Belluzzi O. Kinetic study and numerical reconstruction of A-type current in granule cells of rat cerebellar slices. J Neurophysiol 69: 2222–2231, 1993.
Begenisich T. Conditioning hyperpolarization-induced delays in the potassium channels of myelinated nerve. Biophys J 27: 257–265, 1979.[Web of Science][Medline]
Belluzzi O, Sacchi O, and Wanke E. A fast transient outward current in the rat sympathetic neuron studied under voltage-clamp conditions. J Physiol 358: 91–108, 1985.
Bezanilla F and Armstrong CM. Inactivation of the sodium channel. I. Sodium current experiments. J Gen Physiol 70: 549–566, 1977.
Bilbaut A, Chorvatova A, Ojeda C, and Rougier O. The transient outward current of isolated bovine adrenal zona fasciculata cells: comparison between standard and perforated patch recording methods. J Membr Biol 149: 233–247, 1996.[Web of Science][Medline]
Brew HM and Forsythe ID. Two voltage-dependent K+ conductances with complementary functions in postsynaptic integration at a central auditory synapse. J Neurosci 15: 8011–8022, 1995.[Abstract]
Chandy KG and Gutman GA. Voltage-gated potassium channel genes. In: Ligand and Voltage-Gated Ion Channels, edited by North AR. Boca Raton, FL: CRC, 1994, p. 1–71.
Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, and Rudy B. Molecular diversity of K+ channels. Ann NY Acad Sci 868: 233–285, 1999.[Web of Science][Medline]
Fitzakerley JL, Star KV, Rinn JL, and Elmquist BJ. Expression of Shal potassium channel subunits in the adult and developing cochlear nucleus of the mouse. Hear Res 147: 31–45, 2000.[Web of Science][Medline]
Fonseca R, Hallows J, Trimmer J, Rhodes K, and Tempel B. Localization of Kv channels in the auditory system. Assoc Res Otolaryngol Abstr 21: 213, 1998.
Grigg JJ, Brew HM, and Tempel BL. Differential expression of voltage-gated potassium channel genes in auditory nuclei of the mouse brain stem. Hear Res 140: 77–90, 2000.[Web of Science][Medline]
Grissmer S, Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA, Karmilowicz MJ, Auperin DD, and Chandy KG. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol Pharmacol 45: 1227–1234, 1994.[Abstract]
Heck W, JL F, K B, and L S. Localization of a fast inactivating voltage-gated potassium channel (Kv1.4) in the cochlear nucleus of the adult mouse. Assoc Res Otolaryngol Abstr 20: 82, 1997.
Heinemann SH, Rettig J, Graack HR, and Pongs O. Functional characterization of Kv channel beta-subunits from rat brain. J Physiol 493: 625–633, 1996.
Heinemann S, Rettig J, Scott V, Parcej DN, Lorra C, Dolly J, and Pongs O. The inactivation behaviour of voltage-gated K channels may be determined by association of alpha- and beta-subunits. J Physiol 88: 173–180, 1994.
Hodgkin AL and Huxley AF. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117: 500–544, 1952.
Hopkins WF, Allen ML, Houamed KM, and Tempel BL. Properties of voltage-gated K+ currents expressed in Xenopus oocytes by mKv1.1, mKv1.2 and their heteromultimers as revealed by mutagenesis of the dendrotoxin-binding site in mKv1.1. Pflugers Arch 428: 382–390, 1994.[Web of Science][Medline]
Hsiao CF and Chandler SH. Characteristics of a fast transient outward current in guinea pig trigeminal motoneurons. Brain Res 695: 217–226, 1995.[Web of Science][Medline]
Jonas EA and Kaczmarek LK. Regulation of potassium channels by protein kinases. Curr Opin Neurobiol 6: 318–323, 1996.[Web of Science][Medline]
Kanemasa T, Gan L, Perney TM, Wang LY, and Kaczmarek LK. Electrophysiological and pharmacological characterization of a mammalian Shaw channel expressed in NIH 3T3 fibroblasts. J Neurophysiol 74: 207–217, 1995.
Kanold PO and Manis PB. Transient potassium currents regulate the discharge patterns of dorsal cochlear nucleus pyramidal cells. J Neurosci 19: 2195–2208, 1999.
Koschak A, Bugianesi RM, Mitterdorfer J, Kaczorowski GJ, Garcia ML, and Knaus HG. Subunit composition of brain voltage-gated potassium channels determined by hongotoxin-1, a novel peptide derived from Centruroides limbatus venom. J Biol Chem 273: 2639–2644, 1998.
Kues WA and Wunder F. Heterogeneous expression patterns of mammalian potassium channel genes in developing and adult rat brain. Eur J Neurosci 4: 1296–1308, 1992.[Web of Science][Medline]
Manis PB. Membrane properties and discharge characteristics of guinea pig dorsal cochlear nucleus neurons studied in vitro. J Neurosci 10: 2338–2351, 1990.[Abstract]
Manis PB and Marx SO. Outward currents in isolated ventral cochlear nucleus neurons. J Neurosci 11: 2865–2880, 1991.[Abstract]
Manis PB, Marx SO, and White JA. Integrative mechanisms of ventral cochlear nucleus stellate cells: Membrane conductances and electrotonic structure. In: Cochlear Nucleus: Structure and Function in Relation to Modeling, edited by Ainsworth WA. London: JAI, 1996, p. 213–229.
McCormack K, McCormack T, Tanouye M, Rudy B, and Stuhmer W. Alternative splicing of the human Shaker K+ channel beta 1 gene and functional expression of the beta 2 gene product. FEBS Lett 370: 32–36, 1995.[Web of Science][Medline]
Nagatomo T, Inenaga K, and Yamashita H. Transient outward current in adult rate supraoptic neurons with slice patch-clamp technique: inhibition by angiotensin II. J Physiol 485: 87–96, 1995.
Nakahira K, Shi G, Rhodes KJ, and Trimmer JS. Selective interaction of voltage-gated K+ channel beta-subunits with alpha-subunits. J Biol Chem 271: 7084–7089, 1996.
Oertel D. Synaptic responses and electrical properties of cells in brain slices of the mouse anteroventral cochlear nucleus. J Neurosci 3: 2043–2053, 1983.[Abstract]
Owen DG, Hall A, Stephens G, Stow J, and Robertson B. The relative potencies of dendrotoxins as blockers of the cloned voltage-gated K+ channel, mKv1.1 (MK-1), when stably expressed in Chinese hamster ovary cells. Br J Pharmacol 120: 1029–1034, 1997.[Web of Science][Medline]
Parri HR and Crunelli V. Sodium current in rat and cat thalamocortical neurons: role of a non-inactivating component in tonic and burst firing. J Neurosci 18: 854–867, 1998.
Perney TM and Kaczmarek LK. Localization of a high threshold potassium channel in the rat cochlear nucleus. J Comp Neurol 386: 178–202, 1997.[Web of Science][Medline]
Rathouz M and Trussell L. Characterization of outward currents in neurons of the avian nucleus magnocellularis. J Neurophysiol 80: 2824–2835, 1998.
Rettig J, Heinemann SH, Wunder F, Lorra C, Parcej DN, Dolly JO, and Pongs O. Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit. Nature 369: 289–294, 1994.[Medline]
Reyes AD, Rubel EW, and Spain WJ. Membrane properties underlying the firing of neurons in the avian cochlear nucleus. J Neurosci 14: 5352–5364, 1994.[Abstract]
Rhodes KJ, Strassle BW, Monaghan MM, Bekele-Arcuri Z, Matos MF, and Trimmer JS. Association and colocalization of the Kvbeta1 and Kvbeta2 beta-subunits with Kv1 alpha-subunits in mammalian brain K+ channel complexes. J Neurosci 17: 8246–8258, 1997.
Rizzo MA and Nonner W. Transient K current in the somatic membrane of cultured central neurons of embryonic rat. J Neurophysiol 68: 1708–1719, 1992.
Robertson B, Owen D, Stow J, Butler C, and Newland C. Novel effects of dendrotoxin homologues on subtypes of mammalian Kv1 potassium channels expressed in Xenopus oocytes. FEBS Lett 383: 26–30, 1996.[Web of Science][Medline]
Rothman JS and Manis PB. Differential expression of three distinct potassium currents in the ventral cochlear nucleus. J Neurophysiol 89: 3070–3082, 2003a.
Rothman JS and Manis PB. The roles potassium currents play in regulating the electrical activity of ventral cochlear nucleus neurons. J Neurophysiol 89: 3097–3113, 2003b.
Rudy B. Diversity and ubiquity of K channels. Neuroscience 25: 729–749, 1988.[Web of Science][Medline]
Ruppersberg JP, Stocker M, Pongs O, Heinemann SH, Frank R, and Koenen M. Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation. Nature 352: 711–714, 1991.[Medline]
Sah P, Gibb AJ, and Gage PW. The sodium current underlying action potentials in guinea pig hippocampal CA1 neurons. J Gen Physiol 91: 373–398, 1988.
Sanguinetti MC, Curran ME, Spector PS, and Keating MT. Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia. Proc Natl Acad Sci USA 93: 2208–2212, 1996.
Sarkar SN, Adhikari A, and Sikdar SK. Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line. J Physiol 488: 633–645, 1995.
Schoppa NE and Sigworth FJ. Activation of shaker potassium channels. I. Characterization of voltage-dependent transitions. J Gen Physiol 111: 271–294, 1998.
Schwarz DW and Puil E. Firing properties of spherical bushy cells in the anteroventral cochlear nucleus of the gerbil. Hear Res 114: 127–138, 1997.[Web of Science][Medline]
Selyanko AA, Hadley JK, Wood IC, Abogadie FC, Delmas P, Buckley NJ, London B, and Brown DA. Two types of K(+) channel subunit, Erg1 and KCNQ2/3, contribute to the M-like current in a mammalian neuronal cell. J Neurosci 19: 7742–7756, 1999.
Serodio P, Vega-Saenz de Miera E, and Rudy B. Cloning of a novel component of A-type K+ channels operating at subthreshold potentials with unique expression in heart and brain. J Neurophysiol 75: 2174–2179, 1996.
Shi G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, and Trimmer JS. Beta subunits promote K+ channel surface expression through effects early in biosynthesis. Neuron 16: 843–852, 1996.[Web of Science][Medline]
Shi W, Wymore RS, Wang HS, Pan Z, Cohen IS, McKinnon D, and Dixon JE. Identification of two nervous system-specific members of the erg potassium channel gene family. J Neurosci 17: 9423–9432, 1997.
Smith-Maxwell CJ, Ledwell JL, and Aldrich RW. Uncharged S4 residues and cooperativity in voltage-dependent potassium channel activation. J Gen Physiol 111: 421–439, 1998.
Thornhill WB, Wu MB, Jiang X, Wu X, Morgan PT, and Margiotta JF. Expression of KV1.1 delayed rectifier potassium channels in Lec mutant Chinese hamster ovary cell lines reveals a role for sialidation in channel function. J Biol Chem 271: 19093–19098, 1996.
Trudeau MC, Titus SA, Branchaw JL, Ganetzky B, and Robertson GA. Functional analysis of a mouse brain Elk-type K+ channel. J Neurosci 19: 2906–2918, 1999.
Verma-Kurvari S, Border B, and Joho RH. Regional and cellular expression patterns of four K+ channel mRNAs in the adult rat brain. Brain Res Mol Brain Res 46: 54–62, 1997.[Medline]
Wang H, Kunkel DD, Martin TM, Schwartzkroin PA, and Tempel BL. Heteromultimeric K+ channels in terminal and juxtaparanodal regions of neurons. Nature 365: 75–79, 1993.[Medline]
Wang H, Kunkel DD, Schwartzkroin PA, and Tempel BL. Localization of Kv1.1 and Kv1.2, two K channel proteins, to synaptic terminals, somata, and dendrites in the mouse brain. J Neurosci 14: 4588–4599, 1994.[Abstract]
Wang HS, Pan Z, Shi W, Brown BS, Wymore RS, Cohen IS, Dixon JE, and McKinnon D. KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M channel. Science 282: 1890–1893, 1998.
Wang LY, Gan L, Forsythe ID, and Kaczmarek LK. Contribution of the Kv3.1 potassium channel to high-frequency firing in mouse auditory neurons. J Physiol 509: 183–194, 1998.
Weiser M, Vega-Saenz de Miera E, Kentros C, Moreno H, Franzen L, Hillman D, Baker H, and Rudy B. Differential expression of Shaw-related K+ channels in the rat central nervous system. J Neurosci 14: 949–972, 1994.[Abstract]
Yang WP, Levesque PC, Little WA, Conder ML, Ramakrishnan P, Neubauer MG, and Blanar MA. Functional expression of two KvLQT1-related potassium channels responsible for an inherited idiopathic epilepsy. J Biol Chem 273: 19419–19423, 1998.
Young SH and Moore JW. Potassium ion currents in the crayfish giant axon. Dynamic characteristics. Biophys J 36: 723–733, 1981.[Web of Science][Medline]
Zhang S and Trussell LO. A characterization of excitatory postsynaptic potentials in the avian nucleus magnocellularis. J Neurophysiol 72: 705–718, 1994.
This article has been cited by other articles:
|
|
 |
|
  H. Yang and M. A. Xu-Friedman Impact of Synaptic Depression on Spike Timing at the Endbulb of Held J Neurophysiol, September 1, 2009; 102(3): 1699 - 1710. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R Clay, D. Paydarfar, and D. B Forger A simple modification of the Hodgkin and Huxley equations explains type 3 excitability in squid giant axons J R Soc Interface, December 6, 2008; 5(29): 1421 - 1428. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Kuznetsova, M. H. Higgs, and W. J. Spain Adaptation of Firing Rate and Spike-Timing Precision in the Avian Cochlear Nucleus J. Neurosci., November 12, 2008; 28(46): 11906 - 11915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Curti, L. Gomez, R. Budelli, and A. E. Pereda Subthreshold Sodium Current Underlies Essential Functional Specializations at Primary Auditory Afferents J Neurophysiol, April 1, 2008; 99(4): 1683 - 1699. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-J. Cao, S. Shatadal, and D. Oertel Voltage-Sensitive Conductances of Bushy Cells of the Mammalian Ventral Cochlear Nucleus J Neurophysiol, June 1, 2007; 97(6): 3961 - 3975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Guan, J. C. F. Lee, M. H. Higgs, W. J. Spain, and R. C. Foehring Functional Roles of Kv1 Channels in Neocortical Pyramidal Neurons J Neurophysiol, March 1, 2007; 97(3): 1931 - 1940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Klug and L. O. Trussell Activation and Deactivation of Voltage-Dependent K+ Channels During Synaptically Driven Action Potentials in the MNTB J Neurophysiol, September 1, 2006; 96(3): 1547 - 1555. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Guan, J. C. F. Lee, T. Tkatch, D. J. Surmeier, W. E. Armstrong, and R. C. Foehring Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones J. Physiol., March 1, 2006; 571(2): 371 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Xu-Friedman and W. G. Regehr Dynamic-Clamp Analysis of the Effects of Convergence on Spike Timing. I. Many Synaptic Inputs J Neurophysiol, October 1, 2005; 94(4): 2512 - 2525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang and P. B. Manis Synaptic Transmission at the Cochlear Nucleus Endbulb Synapse During Age-Related Hearing Loss in Mice J Neurophysiol, September 1, 2005; 94(3): 1814 - 1824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. R. Fernandez, W. H. Mehaffey, M. L. Molineux, and R. W. Turner High-Threshold K+ Current Increases Gain by Offsetting a Frequency-Dependent Increase in Low-Threshold K+ Current J. Neurosci., January 12, 2005; 25(2): 363 - 371. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Rothman and P. B. Manis Differential Expression of Three Distinct Potassium Currents in the Ventral Cochlear Nucleus J Neurophysiol, June 1, 2003; 89(6): 3070 - 3082. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Rothman and P. B. Manis The Roles Potassium Currents Play in Regulating the Electrical Activity of Ventral Cochlear Nucleus Neurons J Neurophysiol, June 1, 2003; 89(6): 3097 - 3113. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
TOP
ABSTRACT
INTRODUCTION
) and 8 other Type I-t cells (
,
) inactivation time constants.
