One widely expressed K+ channel, often called the "BK" channel for its "big" single channel conductance, is regulated by intracellular Ca2+ and voltage: at constant voltage, the open probability (Po) increases with [Ca2+]. At constant [Ca2+], Po increases with depolarization. BK channels participate in many physiological processes, including repolarization of the action potential (
The mechanism of BK channel gating is addressed by a recent paper in this journal (
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Ion Channels as Allosteric Proteins |
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Cooperative mechanisms were introduced long ago in the field of enzyme kinetics, notably the self-described "plausible" MWC model (
Many ion channels are multisubunit proteins, containing multiple sensors that somehow work together to regulate the functional state of a single centrally located pore. The analogy to allosteric enzymes is most obvious for ligand-gated channels, which have two or more ligand binding sites (
Members of the P domaincontaining superfamily of ion channels contain either four subunits, or four homologous domains, each coupled to a single pore. These too are allosteric proteins (
Models for cooperative activation of voltage-dependent channels began with
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Most studies are consistent with the assumption of Figure 1 and Figure 2; namely, that all voltage sensors must activate before the channel opens. To begin with, channels typically activate with a sigmoidal delay, but deactivate almost exponentially upon repolarization (
Allosteric coupling to voltage sensor movement is also a plausible mechanism for inactivation (
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BK Channels as Allosteric Proteins |
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Regulation of BK channels is particularly complicated, because there are two fundamental regulators (Ca2+ and voltage) instead of one. One early suggestion was that voltage dependence arose from binding of Ca2+ within the membrane's electrical field (
A general scheme for allosteric activation of BK channels must consider three distinct but coupled processes: voltage sensor activation, Ca2+ binding, and channel opening. If all permutations are considered (04 Ca2+ bound, 04 voltage sensors activated, and the channel either open or closed), there are 5 x 5 x 2 = 50 possible states of the channel (Figure 4). In the diagram, the subscripts and superscript denote the number of activated voltage sensors and the number of bound Ca2+ ions, respectively; 16 of 25 open states are "hidden" by closed states. Even that scheme could easily be extended (
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Enough theory for now. What does the data show? Are all those states really necessary?
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Evidence from Macroscopic Ionic and Gating Currents |
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In native cells, BK channels tend to be intimately coupled to voltage-dependent Ca2+ channels, producing a current that depends in a complex manner on Ca2+ entry and diffusion, as well as on voltage. To study the intrinsic kinetics of BK channels at the macroscopic level, in the absence of Ca2+ channels (and this at a constant [Ca2+]), it has proven useful to work with cloned channels in expression systems (
In apparent contrast to the complexities expected from Figure 4, BK currents change nearly exponentially in response to a voltage step. But the time constants depend on both Ca2+ and voltage (
One key result was that BK channels can open in the effective absence of Ca2+ in response to a sufficiently strong depolarization. Without Ca2+, the BK channel is purely voltage dependent, which simplifies the situation and allows the use of established procedures for analyzing voltage-dependent gating. Without Ca2+, Figure 3 reduces to a simple two-state C0O0 model.
Linear models such as Figure 1 and Figure 2 make a strong prediction: that Po will decrease exponentially at extreme negative voltages, with a steepness depending on the amount of charge moved (
The model was supported by analysis of gating currents (
Linear models (Figure 1 and Figure 2) predict that charge movement precedes channel opening, so the voltage dependence of charge movement (the QV curve) is shifted to more negative voltages compared with channel activation (the GV curve). With Figure 3, some charge movement precedes opening, but channels can open before all the gating charge moves, allowing subsequent charge movement in the OO steps. That can produce a "crossover" of the QV and GV curves, which actually has been reported for BK channels (
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Evidence from Single Channels |
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Their high single-channel conductance has long made BK channels a proving ground for kinetic analysis (
While the Aldrich lab concentrated on BK channel gating without Ca2+,
It is tempting to interpret the multiple closed (or open) states in the
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Lessons for Kinetic Modeling of Ion Channels |
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Why is it so difficult to go from kinetic data to a mechanism? Didn't
Macroscopic ionic currents.
For a two-state CO model, the exponentially relaxing current observed in response to a voltage step contains enough information to fully determine the two parameters of that model, the rate constants for channel opening and closing at that voltage (if the current amplitude can somehow be converted to Po). For models like those of
Gating currents.
These provide complementary information, since voltage-sensitive CC or OO transitions produce gating currents. Some practical issues that limit the usefulness of gating currents for channels in most native cells (current isolation, leak, and capacity subtraction) are less problematic for studies using cloned channels in expression systems (see
Single channels.
In principle, it is straightforward to extract kinetic information from single channel data: fit exponentials to the distribution of open and closed dwell times, and get the number of states and their mean lifetimes. Practically, if the range of open and closed times is large (as for BK channels), an immense amount of data is required to define the kinetics, even under a single condition. The Magleby lab has worked for over a decade to define the kinetics of BK channels over a wide range of voltages and [Ca2+]. Definition of the steady state dwell-time distributions does not, however, establish the connectivity between the states, although "2-D" distributions give additional information (see
Given the strengths and limitations of each approach, it is important to use several. But it is far from trivial to combine information from these fundamentally different measurements (macroscopic ionic and gating currents, single channel currents), usually measured under different conditions (as noted above for ionic and gating currents). Going from kinetic data to a model is not a stereotyped, mechanical procedure, but a complex creative enterprise with ample room for different approaches. It is most comforting in this context that the two labs arrive at the same conclusion about the general structure and connectivity of the kinetic scheme underlying channel gating.
Perhaps it is time for a reminder about the goals of kinetic modelling. One motivation is to operationally define the behavior of a channel, to quantitatively define its role in the electrical behavior of a cell. But a modeler interested in (for example) the role of BK channels in AP repolarization will find little of direct use in the papers discussed here. Clearly, their goal was differentto get at the molecular basis of channel gating and to relate formal kinetic diagrams such as Figure 4 to actual conformational states of the ion channel protein. That explains why the models discussed here are based, at least metaphorically, on what is known about channel structure (e.g., the number of subunits).
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Open Questions |
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Cross sections of Figure 4 seem to work at extreme Ca2+ (high or low). It will be crucial to test whether Figure 4 also can describe the often complex Ca2+ dependence of the BK channel (e.g., Hill coefficients), and the interactions between Ca2+ and voltage.
The discussion so far has considered "the" BK channel. The Magleby lab studied native BK channels in rat skeletal muscle and the Aldrich lab studied cloned mouse BK channels (mSlo) expressed in Xenopus oocytes. Gating of the Drosophila dSlo channel differs from muscle BK channels (
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References |
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