The pH Dependence of mSlo3 Currents
Macroscopic currents resulting from expression of mSlo3 cRNA in Xenopus oocytes are illustrated in . At pH 7.0, voltage steps as positive as +240 mV produce only a small fractional activation of current, while a marked increase in current activation is observed with increases in pH over the range of pH 7.4 through pH 9.0. As pH is increased, currents at a given voltage are more rapidly activated.
Figure 1. Increases in pH result in increased Slo3 current activation. (A) An inside-out patch expressing Slo3 channels was bathed with 0 Ca2+ solutions with pH set to the indicated nominal values. Patches were activated by the indicated voltage protocol. (more ...)
An interesting property of the Slo3 currents is the small amplitude and rapid deactivation of tail currents after repolarization (). At symmetric voltages, the maximal detectable tail current amplitude is markedly reduced relative to the current at positive potentials (see below). Because of the small size and rapidity of the inward current tails, conductance–voltage (G/V) curves at each pH () were typically generated from the peak outward currents recorded at each activation potential. Within each patch, conductances under any condition were normalized to the maximal conductance recorded for the full set of measurements over all pH. An increase in pH allows Slo3 current to be activated at more negative voltages () and there is the beginning of some saturation in G/V curves as pH approaches 8.5. We also observed additional shifts in gating when pH is increased to 9.5 (unpublished data), but such shifts appeared to be somewhat irreversible and patch stability was a problem above pH 9.0. A limiting G/V curve at low pH could not be defined even with voltage steps to +300 mV. For a limited set of patches with more robust outward currents, G/Vs generated either by outward currents or from tail currents were compared (). Tail current G/Vs exhibited more variance in conductance estimates, since conductance was estimated from a single point on a rapidly changing current. However, the general shape and relationship between G/V curves at different pH were similar in both cases.
Figure 2. pH dependence of Slo3 macroscopic conductance. In A, steady-state currents were measured from records as in and converted to conductances. For results from any individual patch, G/V curves were normalized to the maximal conductance observed with (more ...)
For descriptive purposes, single Boltzmann curves (Eq. 1
) were fit to the G
curves at different pH (). The G
curves obtained at pH 7.4–8.0 were best fit with a maximal conductance that was less than that obtained at the highest pH. Increases in pH produced modest negative shifts in Vh
() with little effect on z
over all pH). When Gmax
was constrained to the same maximum for all G
curves, the resulting estimated shift in Vh
was increased, but the fit to the G
curves was poor. Thus, the shape of the G
curves over all pH suggests that, at least over the range of technically achievable command potentials, the same maximal conductance is not reached among different pH. This contrasts to the similarity in limiting conductance that is observed at different [Ca2+
] for Slo1 channels (Cox et al., 1997
). The pH-dependent differences in Slo3 conductance observed at the most positive activation potentials mirror the pH-dependent differences in single channel Po
determined from single channel and variance analysis shown in the associated paper (see Zhang et al. on p. 301
of this issue).
Figure 3. Empirical properties of Slo3 G/V curves. In A, the voltage of half activation (Vh) is plotted as a function of proton concentration. Vh estimates were based on Boltzmann fits (Eq. 1) in which Gmax was not constrained. In B, Gmax obtained from fits of (more ...)
The Fractional Conductance of Slo3 at Negative Potentials Is Appreciable and Relatively Voltage Independent
In our typical activation protocols, we often noticed an appreciable increase in current at voltages of −100 mV or more negative as pH was increased. In the associated paper (Zhang et al., 2006
), estimates of average conductance arising from putative Slo3 openings at negative potentials were made. These estimates suggested that conductance at negative potentials may be as much as 0.1% of the conductance at more positive potentials and that the voltage dependence of conductance at negative potentials may be relatively weak. Here we attempt a macroscopic estimate of Slo3 activation at negative potentials.
To accomplish this, we examined currents at voltages from −300 mV through +300 mV in single sweeps for a limited set of pH (7.0, 7.6, and 8.5). Inspection of current traces indicates that at pH 7.0, there is little, if any, channel activation at negative potentials for the durations of steps that we have used. An example of currents from one experiment is shown in for pH 7.0 and pH 8.5. A maximum of ~4 nA of current was observed in this patch at +300 mV ().
High gain examples of currents at pH 7.0, 7.6 (unpublished data), and 8.5 show that over potentials from −20 through −260 mV, there is appreciably more current at pH 7.6 and 8.5 than at pH 7.0 (). Over the range of −20 through −120 mV, current measured at pH 7.0 scaled linearly with voltage with no indication of any Slo3 channel openings. For the moment, making the assumption that pH does not directly affect leak conductance, the linear leak conductance determined at pH 7.0 was subtracted from currents in pH 7.6 and 8.5 to generate G/V curves for each condition. This procedure resulted in log-scaled G/V curves (e.g., ) that approach a limiting conductance at potentials negative to −100 mV of ~0.8–1% of the maximal conductance. Of most interest, the limiting conductance appears to be relatively voltage independent over the range of −150 to −300 mV.
Figure 6. Voltage dependence of Slo3 conductance at negative potentials. In A, the voltage dependence of leak conductance in inside-out patches from DEPC-injected oocytes is displayed at each of three pH, 7.0, 7.6, and 8.5. The increase in leak conductance with (more ...)
However, we were concerned that increasing pH may result in effects on conductance unrelated to Slo3. We have therefore examined effects of pH on currents in patches from oocytes injected with the diethylpyrocarbonate (DEPC)-buffered solutions used for cRNA injection (). In this case, increasing pH from 7.0 to 8.5 results in an increase in leak current, although the leak current never exhibits the flickery behavior characteristic of the Slo3 channels. For a set of four patches from DEPC-injected oocytes, estimates of leak conductance were made at pH 7.0, 7.6, and 8.5 (). Increasing pH from 7.6 to 8.5 resulted in an approximately twofold increase in a leak conductance, with no indications of any voltage dependence in the pH-dependent increase in conductance.
Because of the presence of the pH-dependent effects on leak conductance, subtraction of pH 7.0 conductances from any Slo3 currents observed at pH 8.5 will overestimate the true Slo3 conductance. We therefore employed an alternative subtraction procedure to make estimates of Slo3 conductance at negative potentials. This is illustrated in in which 10-ms traces obtained from 0 to −240 mV are shown at both pH 7.0 and pH 8.5. Although the records at pH 8.5 do not reveal a clear baseline, we have attempted to make a baseline estimate that would provide an estimate of the minimal amount of Slo3 conductance. The rationale for defining the putative baseline was based on three considerations. First, we assume that the true baseline at pH 8.5 varies linearly with voltage (), i.e., leak conductance is voltage independent. Second, we assume that some of the most positive current values in a given trace reflect excursions to the true baseline. Finally, we assume that the variance around the baseline should be similar to the variance obtained either at pH 7.0 or during steps to 0 mV. Given the rather broad shape of the current amplitude distribution, it seems uncertain whether any points in the distribution reflect actual baseline values. However, the purpose of this procedure is to provide a minimum estimate of the possible Slo3 current levels.
Figure 5. Evaluation of the average Slo3 current magnitude at negative potentials. In A, segments of currents obtained at either pH 7.0 or pH 8.5 are compared over a range of voltages for the patch shown in . Dotted line corresponds to the zero current level, (more ...)
For the example in , the more negative dashed line on each panel indicates the baseline defined for this patch for currents at pH 8.5. This baseline estimate varies linearly with voltage, passes through the more positive current values in the trace, and, as shown in , the distribution of current values positive to this current level exhibit a range similar to the range around the baseline determined at pH 7.0. The standard deviation for single Gaussian fits to current records either at pH 7.0 or at 0 mV at pH 8.5 at 5 kHz bandwidth was ~1.1 pA; 95% of all baseline values will be within ± 2.0 pA of the mean baseline value. For baseline estimates at pH 8.5 shown in , over the range of voltages from −160 through −240 mV, the baseline estimates are >2.0 pA from the most positive current values observed in the tail of the distributions. Thus, the selected baseline seems a conservative estimate of the most negative possible baseline. The mean value of all current in the record corrected for this baseline value should therefore provide a minimal estimate of Slo3 current. However, it is likely that the true baseline will be positive to that selected (although not as positive as the pH 7.0 current level) such that the actual current arising from Slo3 will be intermediate between the two estimates. Assuming that for all patches the pH-dependent changes in leak are voltage independent (as in ), the Slo3 conductances estimated by this procedure should accurately reveal the underlying voltage dependence of Slo3 activation over these potentials.
Based on this methodology, estimates of Slo3 conductance were determined and normalized to the estimates of conductance obtained in the same patches at positive potentials (). The minimum estimates of Slo3 conductance revealed a relatively voltage-independent conductance at negative potentials. An exponential fit of the conductance estimates from −300 through −220 mV yielded a limiting slope of ~0.04 e, while a Boltzmann fit of the values >0 mV yielded z = 0.54 ± 0.012 e. For this same set of patches, the dependence of log(G) on voltage at pH 7.0, 7.6, and 8.5 is shown in , qualitatively revealing that the limiting Gmax differs at each pH.
Because of apparent reductions in single channel current amplitude observed at negative potentials in the associated paper (Zhang et al., 2006
), one might raise a question regarding whether the current estimates at negative potentials can be compared with those at positive potentials. However, we established that the apparent reduction in Slo3 single channel amplitudes at negative potentials almost certainly arises from the effects of filtering and not properties of the conduction process (Zhang et al., 2006
). Therefore, although filtering reduces the apparent single channel amplitude, it does not alter the net current flux passing through any individual channel over time. As a consequence, calculations of conductance based on mean current measurements at negative potentials can be normalized and compared with estimates at positive potentials.
Allosteric Activation of Slo3 Currents
Can regulation of Slo3 by pH be described by models similar to those used to described allosteric regulation of Slo1 channels by Ca2+
? As encapsulated in Scheme 1
, we postulate that both voltage sensor movement and some pH-dependent process independently regulate the closed–open equilibrium. A fundamental difference between regulation of Slo1 and Slo3 is that for Slo3, interaction with the likely regulatory ion, in this case presumably protons, favors channel closure. Therefore, for Slo3, the closed Slo3 conformation corresponds to the liganded channel. As given in Scheme 1
and Eqs. 2
, the formulation requires that protonation be negatively coupled to channel opening, which requires values of C < 1. Also, in contrast to the definition of L
used for Slo1 channels, in the present formulation, since L
defines the C–O equilibrium in the absence of protons, this represents the C–O equilibrium when the cytosolic domain is in its most activated condition.
Despite the limitations of the Slo3 G
curves and the difficulties in estimates of conductance at negative potentials both here for macroscopic currents and for unitary currents in the associated paper (Zhang et al., 2006
), the available information is suitable for defining at least some of the key allosteric constants involved in Slo3 gating. It is important to keep in mind that interpretation of the present macroscopic measurements depends critically on the fact that in the associated paper (Zhang et al., 2006
) we have demonstrated that Slo3 single channel conductance varies ohmically with voltage and is independent of pH. This provides assurance that any pH-dependent effects on macroscopic currents do arise from regulation of the conformational equilibrium rather than from direct effects on ion conductance. Using the estimates of the voltage and pH dependence from the unitary current measurements to renormalize the macroscopic conductance estimates, displays the macroscopic log(G
curves in terms of effective Po
Figure 7. Slo3 steady-state conductance can be described by Scheme 1. In A, the log(Po) vs. voltage relationships at pH 7.0, 7.6, and 8.5 were fit (red line) with Scheme 1, assuming zL = 0.04, and constraining C and E. Macroscopic conductances were converted (more ...)
Estimation of zL, L(0), D, and zJ
At very negative potentials, Slo1 voltage sensors almost exclusively reside in resting positions (Horrigan and Aldrich, 2002
). Hence, at sufficiently negative potentials, changes in conductance and the voltage dependence of conductance reflect primarily the C–O equilibrium and its coupling to the ligand-dependent equilibrium. Although direct measurements of the Slo3 voltage sensor equilibrium are not available, the Slo3 log(G
) vs. V
curve exhibits two regions of distinct slopes, similar to Slo1, characteristic of the L
equilibria. Thus, at the most negative potentials, the residual voltage dependence of activation should reflect the intrinsic voltage dependence of L
. The voltage dependence of conductance under conditions where voltage sensors are largely inactive is described by the following scheme.
Since, at negative potentials and over all pH, the normalized fractional conductance is <0.01, (1 + K
(1 + KC
. Eq. 4
can then be simplified to
Furthermore, at the most elevated pH, (1 + KC
)/(1 + K
) ~ 1 such that
Therefore, the relationship between conductance and voltage at negative potentials and elevated pH allows definition both of L(0) and zL, where L(0) for Slo3 is defined as the closed–open equilibrium with an unliganded cytosolic structure.
The fit of the limiting conductance at negative potentials for Slo3 yielded an estimate of zL
= 0.04 e
. This value corresponds reasonably well with the estimate of voltage dependence of single channel conductance of 0.075 e
over the more positive voltage range of −100 to −200 mV (Zhang et al., 2006
). In accordance with the definition of L
(0) therefore corresponds to the Y intercept of the limiting conductance observed at negative potentials. After conversion of the normalized conductance () to absolute Po
() as determined in the associated paper (Zhang et al., 2006
(0) at pH 8.5 was (1.25 ± 1.0) * 10−3
. Since at pH 8.5, Slo3 activation is somewhat less than maximal, we would expect the true value for L
(0) to be somewhat higher.
Finally, the log(G
) vs. V
relationship provides some information about D
, the coupling factor between voltage sensor movement and channel opening and also zJ
, the voltage dependence of the voltage sensor equilibrium. At elevated pH, channel Po
is given by
For the case that L
has a negligible voltage dependence (i.e., zL
is small) and that the most positive potentials strongly favor voltage sensors in activated states, the ratio between Po
at the most positive and negative potentials is approximated by
For Slo3 at pH 8.5, a change in voltage from −300 to +300 results in an ~250–400-fold increase in channel open probability. This suggests a value for D of ~4–4.5.
Based on the log(Po)/V relationship, the above considerations place important constraints on several of the allosteric factors required to describe Slo3 gating equilibrium. It would also be desirable to define parameters for the protonation equilibrium under limiting conditions, e.g., with voltage sensors either in resting or active conditions. Although the available G/V curves essentially define the foot of the concentration–response curve for the effects of protonation, the absence of a measurable current at low pH means that estimates of the coupling constant between the protonation equilibrium and L will be imprecise.
Using the above considerations to constrain some of the parameters, we used Eq. 2
to fit the log(Po
was constrained to 0.04 e. C
was constrained to 0.125, which is the inverse of the constant coupling Ca2+
binding to L
in Slo1 (Horrigan and Aldrich, 2002
was fixed at 0.7 (corresponding to a value of 1.4 for Slo1). With these constraints, an iterative fitting procedure rapidly converged () and the resulting values for L
(0) ([1.59 ± 0.13] * 10−3
) and D
(4.03 ± 0.14) were in reasonable correspondence with the expected values based on the log(Po
) vs. V
relationship given above. If C
was allowed to vary, iterative fitting did not reach a convergence as C
drifted toward 0. Changes in C
to values other than 0.125 did not improve the ability of the model to approximate the unique characteristics of the Slo3 datasets. Similarly, allowing E
to vary did not result in any improvement in the fits and suggests that effects of pH on the voltage-sensing apparatus itself are unlikely to underlie the pH-mediated effects on Slo3 gating. Somewhat improved fits were obtained if zL
was allowed to vary with the optimal fits occurring as zL
approaches 0. zJ
for Slo3 is somewhat smaller than for Slo1, 0.34 vs. 0.58, and this appears consistent with the shallower macroscopic G
curves observed for Slo3.
Allosteric Constants Comparing Slo3 and Slo3 Activation
Another indication of the voltage dependence of charge movement can be provided by determination of the mean activation charge displacement, qa
(Sigg and Bezanilla, 1997
is calculated from the logarithmic slope of the Po
relationship, and values of qa
at negative potentials provide a direct estimate of the limiting zL
value (Ma et al., 2006
). The qa
relationship for Slo3 is given in , along with the expected qa
relationship based on the fit to the log(Po
relationship. The ability of the set of parameters defined by fitting the log(Po
relationship to describe other aspects of our Slo3 results is also summarized in . In C, a plot of Po
for the data in A shows the adequacy of the fitted curves at the more positive activation potentials. For data shown earlier in from a different set of patches over a wider range of pH, the same parameters also provide a reasonable description of the change in conductance both as a function of voltage and pH (). Finally, the same set of parameters describes the Po
relationship () based on the single channel analysis presented in the associated paper (Zhang et al., 2006
Values for all parameters are given in . Visually, the parameters predict G
curves that capture well the general behavior of the experimentally obtained G
curves, although at lower pH, the shifts in G
curves are less well described. It is possible that secondary effects of lower pH may also influence the G
curves. For example, an activating effect of pH below 7.0 has been observed in Slo1 (Avdonin et al., 2003
). However, a number of conclusions are clear. First, the general allosteric model given in Scheme 1
is a reasonable model for Slo3 activation, suggesting that both a protonation-dependent process and voltage sensor movement independently couple to the Slo3 closed–open conformational change. Second, the two major factors that contribute to the differences in G
curves between Slo1 and Slo3 are, first, that for Slo3, the intrinsic voltage dependence of the C–O equilibrium, defined by zL
, is much weaker and, second, that coupling of voltage sensor movement to channel opening, defined by D
, is weaker.
To illustrate these differences and the impact of changes in zL
, versus changing other parameters, the parameters given in were used to generate the expected log(Po
) vs. V
relationship for pH 7.0, 7.6, and 8.5 () and compared with similar predictions for Slo1 based on parameters from Horrigan and Aldrich (2002)
also given in . Then, the parameters zL
were changed individually (D
, ; zL
, ) or together () to illustrate that these two parameters can account for most of the major differences in behavior between Slo1 and Slo3 G
curves. Changes in other parameters, L
(0), or C
, produce changes that do not account for the differences between Slo1 and Slo3, while the difference in zJ
between Slo1 and Slo3 contributes in part to the shallower G
relation for Slo3.
Figure 8. The primary differences between the voltage dependence of Slo3 and Slo1 conductance arise from differences in zL and D. In A, predicted log(Po /V) relationships for Slo3 based in parameters in for pH 7.0, 7.6, and 8.5 are shown in red, along with (more ...)
Slo3 Activation and Deactivation Kinetics Exhibit Two Exponential Components
We now provide a description of the activation and deactivation behavior of Slo3 gating kinetics.
illustrates the time course of Slo3 current activation and its dependence on pH and depolarization. Traces plotted on a linear time base (left column) are fit with single exponential functions, while middle and righthand columns compare the ability of single or double exponential functions to describe the activation behavior. At both lower (7.4) and higher pH (9.0), activation is reasonably well described by a single exponential function over the full range of activation voltages ( and ). However, over the range of pH (7.6–8.5) that produces the primary shifts in G/V curves, two exponential components are clearly required.
Figure 9. Slo3 activation time course is defined by two exponential components. In A, the Slo3 activation time course is illustrated for pH from 9.0 to 7.4 (top to bottom) at voltages from +40 to +240 mV. Blue lines indicate single exponential fits (more ...)
Figure 10. Changes in Slo3 activation time course is more strongly influenced by pH than voltage. Traces from were normalized and grouped to illustrate the dependence of activation time course either on voltage (A) or pH (B). In A, traces on the top show (more ...)
The two exponential components in the activation time course of Slo3 contrast markedly with the behavior of Slo1 (Zhang et al., 2001
; Horrigan and Aldrich, 2002
). Despite the complexity in Slo1 gating models involving both Ca2+
- and voltage-dependent transitions, for Slo1 it is thought that voltage sensor movement and Ca2+
binding steps are rapid and essentially in equilibrium during the time course of current activation. Thus, the observed kinetic relaxation is thought to reflect the conformational change corresponding to opening of the channel (Zhang et al., 2001
; Horrigan and Aldrich, 2002
To assess qualitatively the contributions of pH- and voltage-dependent processes to the activation time course, currents activated under different conditions were normalized and the activation time courses compared either as a function of voltage () or of pH (). For patches with smaller currents and at lower pH, stochastic fluctuations in currents contributed to variability in estimates of fast and slow time constants and the relative amplitude of each component. However, the overall trends of the behavior of each component were consistent among all patches. An 80-mV change in activation potential produces an ~1.5–2-fold change in the half-current activation time irrespective of the pH (). In contrast, the time of half activation of current shifts approximately an order of magnitude as pH is increased from 7.4 to 9.0, irrespective of the activation voltage (). Qualitatively, this behavior is not unlike that of Slo1. For Slo1, increases in Ca2+
from 1 to 100 μM produce an ~10-fold change in activation time constant at a given potential, while an 80-mV change in activation voltage produces an approximately twofold change in activation time constant (Zhang et al., 2001
). Thus, despite the differences in the number of exponential components required to describe the activation time course, qualitatively the general effects of ligand and voltage on overall rates of activation seem similar between Slo1 and Slo3.
Examination of the specific changes in the fast (τf) and slow (τs) components as a function of changes in voltage reveals that over the range of voltages from 120 to 240 mV, there is an ~1.5-fold decrease in τf, with little clear change in τs (). Similarly, changes in pH are associated with rather negligible changes in either τf or τs. In general, τs ~ 10 ms, while τf ~ 1 ms, and at pH 7.4, the single exponential describing the activation time course appears to be predominantly τs, while at pH 9.0, the single exponential appears to be exclusively τf. At intermediate pH, despite the small changes in the actual values of τs and τf, there is a prominent pH-dependent change in the relative amplitudes of the two time constants (). Whereas at pH 8.0, the fast relaxation contributes ~75% to the total activation time course, at pH 7.6, the fast relaxation contributes only ~40%. Thus, there is a steep dependence on pH of the ratio of the amplitudes of the two components, As/Af, with small effects on the actual time constants themselves. Voltage, per se, has minimal effect on the relative contributions of the two components over the range of +120 to +240 mV (). Thus, the differences in the relative effects of pH and voltage on the amplitude ratios account for the stronger effect of pH than voltage on overall activation rate.
Figure 11. Properties of two components of slo3 activation. In A, fast (red) and slow (blue) time constants of activation at the indicated pH are plotted as a function of activation potential. Error bars are SEM of seven to eight estimates. The single exponential (more ...)
We next evaluated the contribution of the slow and fast current components to the overall absolute conductance. Fitted estimates of the slow and fast amplitude components were converted to conductances and then normalized to the overall maximum conductance observed in a given patch at pH 9.0. For the slow component (), given that pH 7.0 activates very little current, the absolute magnitude of this slow conductance increases from pH 7.0 through pH 7.6, but then decreases at higher pH. Because of its small amplitude, no estimate of the magnitude of the slow component of conductance was made above pH 8.0. In contrast, the fast component increases over all pH () and the sum of the amplitudes of the two current components estimated from fitting the activation time course agrees closely with direct measurements of the steady-state conductance (). Thus, the changes in the ratio of the fast and slow component with increases in pH involve both an increase in the absolute contribution of the fast component and a decrease in absolute magnitude of the slower component.
Deactivation of Slo3 macroscopic currents.
Another unusual feature of the Slo3 current is the small amplitude and rapid time course of the tail current after repolarization to negative potentials. To examine this more closely, mSlo3 current was activated by depolarizations to +200 mV, followed by repolarizations to potentials between −200 and +180 mV at pH from 7.4 to 9.0 (). At potentials negative to 0 mV, with a typical recording bandwidth of 10 kHz, two exponential components were clearly observed in the tail current (; ) with the more rapid component having a time constant of <50 μs. Empirically, the rapid deactivation of Slo3 tail current results in an apparent inward current rectification in the instantaneous current–voltage curve. We tested whether the rapid deactivation might be influenced by particular components of our extracellular solution. In addition to K+, our usual extracellular solutions contained 10 mM HEPES and also 2 mM Mg2+. Extracellular solutions containing either 1 mM HEPES or 1 mM EDTA and no added Mg2+ had no effect on the tail currents (unpublished data).
Figure 12. Slo3 deactivation contains two components. In A, the indicated voltage step protocol was used to examine tail current properties at potentials from −200 to +180 at various pH as indicated. Peak tail current at −200 mV is markedly (more ...)
Figure 13. Dependence of deactivation on voltage and pH. In A, tail currents at pH 8.5 are shown on a linear time scale for −40, −120, and −200 mV. In B, traces from A are shown on a logarithmic time scale along with a two component exponential (more ...)
The general properties of the two components of deactivation are summarized in . Tail currents at different voltages () and pH () were compared on both linear and logarithmic time scales. Fits of a two component exponential are required to describe the current decays. For changes in tail current potential (), the primary change in the tail currents is a decrease in the relative amplitude of the slow component (τs) of tail current at more negative potentials (). For changes in pH at a fixed voltage, the properties of the tail currents are remarkably similar (). Examination of the dependence of the two components on voltage and pH reveals some interesting features. First, at a given pH, voltages from −40 through −200 have little effect on τs, and only weak effects on τf (), although the contribution of the fast component increases at more negative voltages (). Second, at −200 mV, changes in pH from 7.4 to 9.0 have essentially no effect on τf or τs or the ratio between the two components, despite the fact that the outward current varies by at least twofold at the activation potential of +200 mV.
Because both activation and deactivation time course exhibit two exponential components, it is tempting to assume that each component describing the activation time course correlates with one of the two components contributing to deactivation. However, the fast component of deactivation may not be directly related to either of the components seen in the activation time course. Specifically, the fast time constant of current activation and the slow time constant of deactivation have very comparable values and both only exhibit a weak voltage dependence, if any. This suggests that the activation τf
and deactivation τs
may reflect the same underlying process. In contrast, for the fast component of deactivation, there is no obvious correspondence to any measured component of the activation process. Perhaps related to the idea that the fast component of tail current may be unrelated to either of the reported time constants of current activation, the flickery Slo3 single channel behavior (Zhang et al., 2006
) suggests there is a fast component of Slo3 gating that is not rate limiting for the macroscopic activation time course.