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1.
A simple method was used to measure directly sodium and potassium currents underlying the action potential in single nerve fibres of Xenopus laevis. A short rectangular stimulus under current-clamp conditions elicited an action potential which was digitally stored and later used as command when voltageclamping the same fibre. The currents thus obtained nearly reproduced the original rectangular stimulus. Adding first 100 nM TTX and subsequently 100 nM TTX plus 10 mM TEA to the extracellular Ringer solution revealed the sodium and the potassium currents during an action potential. They were converted to permeabilities by use of the constant-field equation and are in good agreement with the curves which had been calculated from conventional voltage-clamp data. Thus experimentally determined currents and permeabilities are shown as they are changing during an action potential. 相似文献
2.
Zona-free mouse eggs are amenable to patch recording as well as to whole-cell recording (Bland et al., 1984; Peres, 1986a). Although many patches are silent, others show spontaneous channel opening; the single channel current is 1.5 pA and inwardly directed at resting potential (RP). Current amplitudes distribute normally as a single population at RP, however at RP-50 mV the amplitude histogram indicates two channel populations. Open time distribution is exponential with a mean open time between 4 and 7 msec at RP. In three cases out of thirty-eight, in which depolarizations were given from a holding potential of RP-50 mV, microscopic currents very similar in kinetics and voltage-dependence to the whole-cell Ca2+ current were observed. No single-channel currents could be resolved in these traces. The results reported here indicate that the voltage-dependent Ca2+ current of the mouse egg goes through low-conductance channels located in high density spots on the egg surface. 相似文献
3.
Jon T. Sack Nicholas Stephanopoulos Daniel C. Austin Matthew B. Francis James S. Trimmer 《The Journal of general physiology》2013,142(3):315-324
A family of 40 mammalian voltage-gated potassium (Kv) channels control membrane excitability in electrically excitable cells. The contribution of individual Kv channel types to electrophysiological signaling has been difficult to assign, as few selective inhibitors exist for individual Kv subunits. Guided by the exquisite selectivity of immune system interactions, we find potential for antibody conjugates as selective Kv inhibitors. Here, functionally benign anti-Kv channel monoclonal antibodies (mAbs) were chemically modified to facilitate photoablation of K currents. Antibodies were conjugated to porphyrin compounds that upon photostimulation inflict localized oxidative damage. Anti-Kv4.2 mAb–porphyrin conjugates facilitated photoablation of Kv4.2 currents. The degree of K current ablation was dependent on photon dose and conjugate concentration. Kv channel photoablation was selective for Kv4.2 over Kv4.3 or Kv2.1, yielding specificity not present in existing neurotoxins or other Kv channel inhibitors. We conclude that antibody–porphyrin conjugates are capable of selective photoablation of Kv currents. These findings demonstrate that subtype-specific mAbs that in themselves do not modulate ion channel function are capable of delivering functional payloads to specific ion channel targets. 相似文献
4.
The electrical properties of unfertilized eggs of Fucus serratus L. were characterized using voltage clamp and current clamp with single electrodes. The plasma membrane of the unfertilized egg is excitable. Depolarizing the egg in current clamp induced a transient depolarizing voltage response, the amplitude of which was dependent on the presence of external Ca2+ or Ba2+ and was blocked by La3+. The repolarizing phase was blocked by tetraethylammonium ions. Repeated stimulation at frequencies greater than 0.5 Hz caused a transient loss of excitability. Voltage-clamp experiments revealed that an inward current with an activation threshold of -35 mV underlies the depolarizing phase of the voltage response. This current showed rapid activation and slow inactivation. The current was blocked by La3+ and could be carried by Ca2+ and Ba2+ but not by Sr2+ or Na+. Further depolarization to values more positive than-5 mV induced a slowly activating outward K+ current in addition to the inward current, which corresponded to the repolarizing phase of the voltage response. This K+ current showed little or no inactivation during stimulation and slow deactivation on return to the resting potential. Hyperpolarizing the egg elicited an inward current. On fertilization, the Fucus egg generates a depolarizing fertilization potential. Voltage-clamp experiments revealed an inward fertilization current underlying the fertilization potential. Within 15 min of fertilization a dramatic, irreversible increase in resting K+ permeability developed. The roles of the plasma-membrane channels in generation of the fertilization potential and egg activation are discussed.Abbreviations and Symbols ASW
artificial seawater
- SECC
single-electrode current clamp
- SEVC
single-electrode voltage clamp
- TEA
tetraethylammonium
- Vm
membrane potential
This work was supported by The Marine Biological Association U.K., Science and Engineering Research Council U.K. and The Royal Society of London. 相似文献
5.
Voltage-clamped membrane currents have been investigated from whole-cell patch-clamp recordings performed on single Leydig cells isolated from the adult rat testis. Two outward membrane currents were evoked by depolarizing voltage steps. A potassium current was recorded in cells dialyzed with low (10(-9)-10(-8) M) calcium media. This current was decreased by TEA (10 mM). A chloride current was recorded in cells dialyzed with high (10(-7)-10(-6) M) calcium media. This current was decreased by an external exposure to glutamate. Comparison of the currents at low and high internal calcium concentrations suggests that an increase of the intracellular calcium activates a chloride current. 相似文献
6.
Patch and whole cell calcium currents recorded simultaneously in snail neurons 总被引:4,自引:6,他引:4
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The flow of Ca ions through single Ca channels has been examined. The gigaseal method was used on identifiable snail neurons that were voltage clamped using a two-microelectrode voltage clamp method. Average Ca patch currents and whole cell currents have similar time courses. They are affected similarly by changes in temperature. The differences in amplitude and inactivation between Ba and Ca whole cell currents were present in the patch records. The stationary noise spectra recorded from ensembles of multichannel patches have two components with fast and slow time constants equivalent to two components in the whole cell tail current relaxations. Elementary current amplitudes measured from the variance-mean relationship and from noise spectra gave values comparable to measurements from single channels. The single channel I-V relationship was curvilinear and the maximum slope conductance in 40 mM Cao was 7 pS. The amplitude of unitary currents was unchanged at long times when inactivation had occurred; hence depletion is not involved in this process. Channel density was approximately 3 microns-2 and was the same for Ba and Ca currents. The whole cell asymmetry currents gave very large values for the gating charge per channel. Changes in temperature from 29 to 9 degrees C had only a slight effect on the two Ca tail current tau's at potentials where turn-on of patch and whole cell currents was markedly slowed and the peak amplitudes were reduced by one-third. Single channel recordings were obtained at these two temperatures, and the mean open time and the fast component of the closed times were scarcely affected. Unit amplitudes were reduced by 30% and the slow closed time component was doubled. Therefore, peak currents and the slow closed time component was doubled. Therefore, peak currents were reduced partly as a result of the reduction in unit amplitude, but mainly as a result of a reduction in opening probability, the latter arising from an increase of the long closed times. It is concluded that the behavior of single Ca channels in membrane patches is the same as it is in whole cells. Cooling from 29 to 9 degrees C acts primarily on transitions among closed states and has little effect on the open to closed transition. 相似文献
7.
Frank T. Horrigan 《The Journal of general physiology》2012,140(6):625-634
Large conductance calcium- and voltage-dependent BK potassium channels (aka BKCa, MaxiK, Slo1, KCa1.1, and KCNMA1) are expressed in a wide variety of tissues throughout the body and are activated by both intracellular Ca2+ and membrane depolarization. Owing to these properties, BK channels participate in diverse physiological processes from electrical excitability in neurons and secretory cells, and regulation of smooth muscle tone to tuning of auditory hair cells (Vergara et al., 1998; Ghatta et al., 2006). The response to voltage and Ca2+ allows BK channels to integrate electrical and calcium signaling, which is central to their physiological role. Understanding how BK and other multimodal channels are regulated by and integrate diverse stimuli is not only physiologically important but also relevant to the topic of conformational coupling. As a voltage- and ligand-dependent channel, BK channels contain both voltage-sensor and ligand-binding domains as well as a gate to regulate the flow of K+ through the pore. Coupling of conformational changes in one domain to another provides the basis for transducing voltage and ligand binding into channel opening and, therefore, defines, together with the functional properties of the gate and sensors, the signal transduction properties of the channel. The goal of this perspective is to provide an overview on the role and molecular basis of conformational coupling between functional domains in BK channels and outline some of the questions that remain to be answered.
BK channel structure
The BK channel is a member of the superfamily of voltage-gated channels that assembles as a homotetramer of pore-forming Slo1 (α) subunits. Each subunit contains seven transmembrane segments (S0–S6), including an S5–S6 pore gate domain (PGD) and voltage-sensor domain (VSD) that is likely to include S0–S4 segments (Liu et al., 2008) with charged voltage-sensing residues in S2, S3, and S4 (Fig. 1 A; Ma et al., 2006). In addition, a large C-terminal cytoplasmic domain (CTD) consisting of two homologous regulator of K+ conductance domains (RCK1 and RCK2) contains binding sites for Ca2+ and other ligands (Hou et al., 2009). The CTDs form a tetrameric gating-ring structure whose conformation changes upon Ca2+ binding. Crystal structures of the isolated BK channel gating ring and related prokaryotic Ca2+-activated K+ channel MthK have been solved in the presence and absence of Ca2+ (Jiang et al., 2002; Ye et al., 2006; Wu et al., 2010; Yuan et al., 2010, 2012). The atomic structure of the transmembrane domain has yet to be determined but is assumed to be homologous with that of voltage-gated potassium (KV) channels, consistent with a low resolution cryo-EM structure of the entire channel(Wang and Sigworth, 2009). Although BK channels formed from Slo1 alone are fully functional, each channel may also coassemble with up to four regulatory subunits of which several subtypes exist (β1–4 and γ1–4; Brenner et al., 2000; Yan and Aldrich, 2012). Regulatory subunits tune BK channel function in different tissues, contain one or two transmembrane segments, occupy sites adjacent to the VSD (Liu et al., 2010), and act in part to regulate voltage-dependent gating (Bao and Cox, 2005; Yan and Aldrich, 2010).Open in a separate windowFigure 1.BK channel structure. (A) Topology of the pore-forming Slo1 subunit, including VSD, PGD, and CTD. Charged residues in the VSD that are important for voltage sensing are highlighted in yellow. The CTD contains binding sites for Ca2+, Mg2+, and heme. (B) Schematic organization of functional domains in the tetrameric channel. S6 segments in the PDG are connected to the CTD gating ring through S6-RCK1 linkers.BK channel function
The response of BK channels to voltage and Ca2+ is illustrated in Fig. 2 by plotting steady-state open probability (PO; Fig. 2 A) and Log(PO) (Fig. 2 B) versus voltage at different [Ca2+]i (0–100 µM) for heterologously expressed Slo1 channels (Horrigan and Aldrich, 2002). The Po-V relation in 0 Ca2+ (∼0.5 nM) shows channels can be fully activated in the absence of Ca2+ binding, but only at voltages approaching +300 mV. Calcium, to a first approximation, shifts Po-V to more negative voltages (Fig. 2 A), allowing the channel to activate in a physiological voltage range. However, plotting the data on a log scale reveals that Ca2+ does not simply shift the curve but rather increases Log(PO) in a nearly voltage-independent manner until PO saturates (Fig. 2 B). This response indicates that BK channels are activated independently by voltage- and Ca2+-sensors. Furthermore, Log(PO) becomes almost voltage independent at extreme negative voltages indicating that channels can open in the absence of voltage-sensor activation, a conclusion supported by gating current measurements (Horrigan and Aldrich, 1999).Open in a separate windowFigure 2.Voltage- and Ca2+-gating of BK channels. (A) PO-V relations for mSlo1 estimated as GK/GKmax from macroscopic tail currents after 30 ms voltage pulses in 0–100 µM Ca2+. (B) Log(Po)-V relations extend PO to <10−2 using steady-state unitary current recordings from macropatches. A and B represent mean ± SEM and are fit (solid curves) by the HA model (Horrigan and Aldrich, 2002). The increase in Log(Po) from 0 Ca2+ to saturating 100 µM Ca2+ at −120 mV (where voltage-sensors are in the resting state) reflects Ca2+-sensor/gate coupling energy (). The increase in Log(Po) in 0 Ca2+ from −120 mV to ∼+300 mV (where voltage sensors are fully activated) reflects voltage-sensor/gate coupling (). The values of and are determined from the change in the free energy of the gate (e.g., , where ΔGCO = kTln[PO/(1 ? PO)]), and, in the case of , the measurement at −120 mV must be extrapolated to positive voltages (dashed line) to correct for the weak voltage dependence of the C-O transition (zL = 0.3 e). (C) Allosteric model indicates the possible conformations of the gate (C and O), voltage sensors (R and A), and Ca2+ sensors (X and X-Ca2+) in each of four subunits and allosteric factors (C, D, and E) that describe the energetic coupling between these three parts of the channel. J and L are voltage-dependent equilibrium constants with zero-voltage values J0 and L0 and partial charges (zJ, zL), K = [Ca2+]/KD, where KD is the elementary Ca2+ dissociation constant for the closed channel.An allosteric mechanism of BK channel gating
That BK channels exhibit basal activity in the absence of voltage-sensor activation and Ca2+ binding implies that sensor/gate coupling is not an obligatory process. That is, sensor activation promotes but is not required for channel opening. The ability of voltage or Ca2+ sensors in different subunits to influence a concerted conformational change (opening) in a nonobligatory fashion is well described in terms of allosteric mechanisms (Monod et al., 1965). A dual allosteric model (Fig. 2 C, HA model) was used to fit the steady-state data in Fig. 2 (A and B, curves), accounts for many other features of Slo1 gating (Horrigan and Aldrich, 1999, 2002; Horrigan et al., 1999), and provides a useful framework for analyzing BK channel gating in terms of domain/domain interactions. The HA (Horrigan-Aldrich) model asserts that the channel gate can undergo a closed to open (C-O) conformational change that is regulated by four independent and identical voltage- and Ca2+-sensors. Voltage sensors can be in a resting (R) or activated (A) conformation, whereas Ca2+ sensors can be Ca2+ free (X) or Ca2+ bound (X-Ca2+). The function of each domain is defined by equilibrium constants for gate opening (L), voltage-sensor activation (J), and Ca2+ binding (K). The coupling, or energy transfer, between domains is represented by allosteric factors (C, D, and E) which define the ability of a transition in one domain to affect the equilibrium constant in another.The energetics of conformational coupling
The structures of BK and homologous channels provide important clues concerning the molecular basis of conformational coupling, as discussed below. However, many features of coupling can only be resolved through structure-function analysis using site-directed mutagenesis. A prerequisite to such analysis is to quantify coupling interactions represented by allosteric factors C, D, and E in the HA model. Mutations that alter channel activity may perturb sensors, the gate, or their coupling. Therefore, it is crucial to distinguish changes in coupling from changes in the function of sensor or gate. One way to do this is by fitting steady-state data over a wide range of voltage and [Ca2+] as in Fig. 2 (A and B) to determine all parameters in the HA model. However, a more direct and model-independent approach to measure coupling is to determine the energetic effect on one domain of forcing coupled domains into defined conformations (e.g., all activated or deactivated) under extreme stimulus conditions (see also Chowdhury and Chanda in this issue). For example, the total coupling between all Ca2+ sensors and the gate can be determined by comparing Log(Po) in 0 Ca2+ and saturating 100 µM Ca2+ at extreme negative voltages where voltage sensors are in the resting state, and voltage-sensor/gate coupling can be determined by comparing Log(Po) at extreme negative and positive voltages in 0 Ca2+ (Fig. 2 B; Horrigan and Aldrich, 2002). In general, the HA model predicts that channels can occupy many different open and closed states, defined by the number of voltage- and Ca2+-sensors activated in each channel. But under extreme stimulus conditions, where all voltage- or Ca2+-sensors are either activated or deactivated, gating reduces to a single closed and open state. For a two-state process, the free energy difference between closed and open (ΔGCO) can be defined in terms of the equilibrium constant (Po/1-Po) for the C-O transition (i.e., ΔGCO = kTln[PO/(1 ? PO)]; Chowdhury and Chanda, 2010, 2012). Thus, changes in Log(Po) in Fig. 2 B reflect the energetic effects of voltage- and Ca2+-sensor activation on the gate. Similarly, a weak coupling between voltage- and Ca2+-sensors has been measured by comparing the effects of 0 Ca2+ and saturating Ca2+ on voltage-sensor activation measured with gating currents while the gate is closed (Horrigan and Aldrich, 2002).Although measurement of Ca2+-sensor/gate coupling as in Fig. 2 B is relatively straightforward, measurement of voltage-sensor/gate coupling is subject to several challenges (Fig. 3). The relationship between gating and voltage-sensor/gate coupling is illustrated in Fig. 3 A by expanding the HA model to show the four combinations of states that the gate (C and O) and voltage-sensor (R and A) can assume in a single subunit (RC, RO, AC, and AO) together with the equilibrium constants between them. The equilibrium constant for the C-O transition increases from L when the voltage sensor is in the resting state (Fig. 3 A, #1) to LD when a voltage sensor is activated (Fig. 3 A, #2). Thus the voltage-sensor/gate coupling factor D can be determined by comparing Po at extreme voltages (Fig. 3 B, #1 and 2), with the expectation that Po/(1-Po) will increase by a factor of D4 when all four voltage sensors are activated. One challenge is the large dynamic range of these measurements, which span a seven-order-of-magnitude change in Po and require both macroscopic and unitary current recordings in the same patch and a high level of expression that cannot always be achieved with mutant channels. A related challenge is that Po in 0 Ca2+ approaches saturation near unity at +300 mV as voltage sensors become fully activated (Fig. 2 A). As Po approaches unity, determination of the equilibrium constant Po/(1-Po) becomes problematic and coupling energy can therefore be underestimated, especially in the presence of Ca2+ or with mutants that are easier to open than the WT because Po may saturate before voltage sensors are fully activated. Finally, the C-O conformational change, as in many ion channels, has a weak intrinsic voltage dependence (L(V)) that must be taken into account when comparing Po at extreme voltages (Figs. 2 B and 3 B, dashed lines).Open in a separate windowFigure 3.The energetics of voltage sensor/gate coupling. (A) Voltage sensor/gate states in a single subunit. Numbers refer to data in B, C, and D used to determine the indicated equilibrium constants. (B) PO-V determines L (1) and LD4 (2). Dashed lines indicate the predicted voltage dependence of PO with all voltage sensors either in the resting or activated state and zL = 0.3 e. (C) Normalized QC-V for closed channels determined from ON gating currents (inset) is fit by a Boltzmann function with zJ = 0.58 e and defines J (3). (D) QO-V relation for open channels defines JD (4). QO is estimated by fitting the foot of the qa-V relation (qa = kTdln(PO)/dV) with a Boltzmann function. The shift between QO and QC relations defines the coupling factor D.A complementary approach to measure voltage-sensor/gate coupling is to determine the effect of channel opening on voltage-sensor activation. The equilibrium constant for the R-A transition increases from J when channels are closed to JD when channels are open (Fig. 3 A, #3 and 4). The equilibrium constant J is determined from the charge distribution for closed channels (QC-V; Fig. 3 C). Qc can be measured by integrating ON gating currents (Fig. 3 B, inset) because voltage-sensor activation in BK channels is rapid and occurs while most channels remain closed. The equilibrium constant JD is determined from the charge distribution for open channels (QO-V; Fig. 3 D). QO cannot be determined from gating currents because BK channels close rapidly. However, QO can be estimated in a model-independent fashion from the log-slope of the Po-V relation (qa = kTdln(PO)/dV), as shown in Fig. 3 D (Horrigan and Aldrich, 2002). The QO-V and QC-V relations have the same shape but are shifted relative to each other by a voltage directly proportional to coupling energy for a single voltage sensor (; Ma et al., 2006). This method yields similar results as in Fig. 3 B and avoids challenges relating to the voltage dependence of L(V), or PO saturation. However, gating currents are difficult to measure in BK channels and require an even higher level of expression than in Fig. 3 B.Molecular mechanisms of conformational coupling
Although the energetics and biophysical mechanisms of sensor/gate and sensor/sensor coupling in BK channels are well defined, many fundamental questions remain concerning the molecular basis of these interactions. Where in the channel do they occur? What are the identity and nature of amino acid interactions involved? When do the interactions occur during channel activation? These questions are broad because many potential sites of domain/domain interaction exist, the conformational changes that these domains and their interfaces undergo during gating are not all understood in detail, and insight provided by the structure of BK or homologous channels is in many cases limited. For example, although voltage-sensor/gate coupling is likely to be mediated in large part by interfaces between the VSD and PGD as in Kv channels, there is also a unique interface between the VSD and CTD in BK channels that may contribute to this process. Intracellular Mg2+ is a BK channel activator that is coordinated by residues in both VSD and CTD and interacts electrostatically with the voltage sensor, indicating (together with cross-linking experiments) that these domains come in contact (Yang et al., 2007, 2008). The primary functional effect of Mg2+ is to enhance voltage-sensor/gate coupling, suggesting that VSD/CTD interaction could provide a basis for this process (Horrigan and Ma, 2008). VSD/CTD interaction must also mediate coupling between voltage and Ca2+ sensors. However information about the VSD/CTD interface is incomplete. Structures of BK and MthK channel CTDs and the Kv channel VSD help identifying residues that may lie at this interface. But the complete BK channel structure including the VSD/CTD interface has yet to be determined, and neither MthK nor Kv channels contain a homologous interface.Because most domain/domain interactions involved in conformational coupling remain to be identified, it is useful to consider what properties such interactions must have. In general, coupling may occur through linkers that directly connect two domains or through noncovalent interactions at domain/domain interfaces. In BK channels, Ca2+-sensor/gate coupling is thought to be mediated by the S6-RCK1 linker connecting CTD to PGD (Jiang et al., 2002; Niu et al., 2004). However multiple domain/domain interfaces are also involved in coupling, as noted above. Structural information has helped to identify where these interfaces are and to locate potential interaction partners. But elucidating the basis of conformational coupling is more complex than simply identifying domain/domain interactions. Coupling is necessarily a state-dependent process that depends on the conformations of two coupled domains. To understand the possible interaction mechanisms, it is useful to consider the energetics of coupling in the context of a gating cycle such as that describing voltage-sensor/gate coupling in Fig. 3 A. The coupling energy between a single voltage sensor and gate can be expressed in terms of the free energies of the four states involved (AC, AO, RC, and RO):(1)Therefore, the change in coupling produced by changes in the free energy of each state is:(2)Eq. 2 indicates that coupling is enhanced () by decreasing or (stabilizing the RC or AO state) or by increasing or (destabilizing the AC or RO state). Coupling could also be enhanced by changing the free energy of multiple states, provided the net contribution (Eq. 2) is positive. However, interactions that are state independent or depend on the conformation of only one domain (e.g., or ) will have no effect on coupling (i.e.,).This analysis has several important implications for elucidating mechanisms of conformational coupling. First, to identify coupling interactions by structural methods requires that structures be solved in four conformations defined by two domains and have sufficient resolution to observe state-dependent changes in interaction. In practice, because changes in interaction energy may be subtle or difficult to estimate based on structure, coupling interactions would have to be verified and quantified by structure-function analysis. When only one or two structures of a channel are available, as is currently the case for BK and homologous channels, then structure-function analysis is required to determine if observed interactions participate in coupling, and residues that do not interact in an observed structure may still mediate coupling by interacting in other conformations. Second, structure-function analysis involving site-directed mutagenesis and measurements of coupling energy, as in Figs. 2 and and3,3, should be sufficient to identify residues involved in coupling interactions. However, such experiments must be interpreted cautiously. Allosteric proteins are often sensitive to modulation at domain interfaces by heterotropic ligands that bind and introduce interactions that do not normally exist, and mutations can have similar effects. For example, introduction of a positively charged residue near the BK channel Mg2+-binding site (Q397R) mimics the effect of Mg2+ by introducing an electrostatic interaction (Yang et al., 2007). Thus, the ability of Q397R to enhance coupling does not indicate that Q397 is normally involved in this process, just that it is located at a sensitive interface where conformational changes associated with voltage-sensor activation and channel opening both occur. Such false positives cannot be avoided simply by restricting analysis to mutations that reduce coupling because, based on Eq. 2, coupling can be reduced by introducing interactions that destabilize the RC or AO states or stabilize the AC or RO states. Intracellular heme, which inhibits voltage-sensor/gate coupling in BK channels, may act in this way (Horrigan et al., 2005). Consequently, whether a mutation increases or decreases coupling is less critical than the use of mutations such as Ala that are least likely to introduce interactions. Finally, to define the mechanism of voltage-sensor/gate coupling we must not only identify mutations that alter coupling but also characterize those changes in terms of the effect on individual states (i.e., RC, RO, AC, and AO) to determine when during activation the interactions occur. Although the change in free energy of any particular state cannot be determined directly, the equilibria between states can be evaluated as in Fig. 3 to define the change in free energy of individual states relative to each other. Such analysis has indicated that Mg2+ enhances voltage-sensor/gate coupling by preferentially stabilizing the AO state (Horrigan and Ma, 2008), whereas a stabilization of the RO state is consistent with the inhibitory effect of heme (Horrigan et al., 2005). It is worth noting that, depending on their state dependence, coupling interactions may influence open probability when sensors are in a resting conformation. Thus, the basal activity of BK channels in the absence of sensor activation defined by the equilibrium constant L in the HA model cannot be interpreted simply as the intrinsic stability of the gate because it also reflects any state-dependent interactions between the gate and resting sensors.Mechanisms of voltage-sensor/gate coupling
Kv channel structures reveal two interfaces between VSD and PGD: intrasubunit contacts between the S4–S5 linker and S6 and intersubunit contacts between S4 and S5 (Long et al., 2005a, 2007). In BK channels, additional intersubunit interactions occur between the VSD and CTD (Yang et al., 2008). All of these interfaces potentially play a role in voltage-sensor/gate coupling in BK channels, but their relative contributions and mechanisms remain unknown. The S4/S5 interface is implicated because a mutation near the top of S4 (R210E) reduces coupling energy by half (Ma et al., 2006). However, we cannot rule out that this is a false positive produced by introducing rather than disrupting an interaction because other mutations at this site (R210C and R210N) appear to constitutively activate the channel preventing measurement of voltage-sensor/gate coupling (Ma et al., 2006). Similarly, the VSD/CTD interface is implicated indirectly based on the ability of Mg2+, Q397R, and heme to modulate coupling by introducing interactions at or near this interface.Interactions between the S4–S5 linker and S6 are widely considered to underlie voltage-sensor/gate coupling, also known as electromechanical coupling, in Kv channels (Lu et al., 2002; Tristani-Firouzi et al., 2002; Long et al., 2005b; Chowdhury and Chanda, 2012) and are also likely to be important in BK channels (Sun et al., 2012). However, even in Kv channels many of the questions raised above remain to be answered, such as what are the individual residues and nature of interactions that contribute to coupling and when do they occur during gating? The importance of these questions can be illustrated by comparing three hypothetical mechanisms of voltage-sensor/gate coupling that include S4–S5/S6 interactions and are consistent with the allosteric nature of voltage gating in Slo1 (Fig. 4). Fig. 4 (A–C) depicts the four combinations of states the gate and voltage-sensor can assume in a single subunit. The possibility that S4–S5/S6 forms a rigid connection that forces sensor and gate to move as a unit can be ruled out because voltage sensors can activate while channels remain closed. However, it is conceivable that the S4–S5 linker remains bound to S6 at all times, whereas flexibility in adjoining regions allows sensor and gate to move as if coupled by a spring (Fig. 4 A). Another possibility is the S4–S5 linker binds to the open gate only when the voltage sensor is activated, stabilizing the AO state (Fig. 4 B). Alternatively, the S4–S5 linker of the resting voltage sensor might clash with the open gate to destabilize the RO state (Fig. 4 C). These and other mechanisms could account for the ability of voltage-sensor activation to promote opening but make different predictions concerning the source of coupling and the role of S4–S5/S6 interaction. Fig. 4 A suggests that many parts of the S4–S5 linker could contribute to coupling by influencing the mechanical properties of the linkage. In this case, S4–S5/S6 interaction acts merely as a passive connection that affects coupling only to the extent that it is intact or disrupted. In contrast, Fig. 4 (B and C) predicts that the S4–S5/S6 interaction energy is the main determinant of coupling energy. However, the panels in Fig. 4 differ in the nature of the proposed interaction (Fig. 4 B, binding; Fig. 4 C, steric hindrance) and the state dependence of the interaction. The analysis of coupling energy and equilibria for such gating cycles, as outlined above, should allow such mechanisms and predictions to be distinguished in BK channels.Open in a separate windowFigure 4.Three models of voltage-sensor/gate coupling. Interaction of S4–S5 linker with the S6 gate in a single subunit. The voltage sensor can be in a resting (R) or activated (A) state, and the gate is closed (C) or open (O). (A) Flexible linkage with S4–S5/S6 interacting in all states. (B) S4–S5/S6 binding stabilizes AO state. (C) Steric hindrance destabilizes the RO state.Despite substantial homology between BK and KV channels, important differences also exist that could be relevant to voltage-sensor/gate coupling. First, BK channels are weakly voltage dependent compared with Kv channels and exhibit a different pattern and contribution of voltage-sensing residues in the VSD, suggesting that conformational changes associated with voltage-sensor activation may differ (Ma et al., 2006). Second, the ability of intracellular blockers and Cys-modifying reagents to access the inner pore of closed BK channels (Wilkens and Aldrich, 2006; Zhou et al., 2011) suggests that the BK channel gate may be formed by the selectivity filter (Cox and Hoshi, 2011; Thompson and Begenisich, 2012), as in cyclic-nucleotide–gated channels (Contreras et al., 2008), rather than by crossing of S6 segments at the inner mouth of the pore, as in Kv channels (del Camino and Yellen, 2001). These differences do not require that voltage-sensor/gate coupling occurs through fundamentally different mechanisms. For example, if the BK channel gate is in the selectivity filter it is still likely to be strongly coupled to S6 movement, as in CNG channels (Flynn and Zagotta, 2001), and therefore subject to control by S4–S5/S6 interaction. Indeed, there is considerable evidence for S6 movement associated with BK channel opening (Li and Aldrich, 2006; Wu et al., 2009; Chen and Aldrich, 2011). However, the coupling mechanisms for BK and Kv channels may differ in detail, and a selectivity gate in the BK channel could potentially support a larger role for S4/S5 interaction in voltage-sensor/gate coupling. It should also be noted that regulatory β and γ subunits have been reported to modulate voltage-sensor/gate coupling (Bao and Cox, 2005; Yan and Aldrich, 2010); thus, additional state-dependent interactions may form in BK channels between the VSD and regulatory subunits.Mechanisms of coupling Ca2+ sensors to the gate and voltage sensors
Of the three coupling interactions in the HA model, Ca2+-sensor/gate coupling is easiest to measure and best understood at a molecular level. Nonetheless, many questions and controversies remain to be resolved concerning the conformational changes that occur upon Ca2+ binding and the mechanisms coupling these changes to the gate and voltage sensors.Ca2+-dependent activation is generally consistent with the idea, originally proposed for MthK, that Ca2+ binding causes a conformational change in the gating ring that opens the channel by pulling on the RCK1-S6 linker (Jiang et al., 2002). Crystal structures in the presence and absence of Ca2+ suggest that MthK and BK channel gating rings expand in diameter by 8 and 12 Å, respectively, upon Ca2+ binding (Ye et al., 2006; Wu et al., 2010; Yuan et al., 2012). Experimental evidence in BK channels shows a monotonic relationship between channel activation and linker length, suggesting that the linker is under constant tension in the presence or absence of Ca2+, like a spring (Niu et al., 2004). Likewise, effects on Ca2+-sensor/gate coupling and Ca2 sensitivity of mutations in the N-terminal AC region of the BK channel RCK1 domain suggest that the flexibility of this region is important for transmitting Ca2+-dependent conformational changes to the gate (Yang et al., 2010). Although the latter results are consistent with the linker hypothesis, they also have raised the possibility that conformational changes in the AC region could be coupled to the gate through direct contact with the PGD. Consistent with this possibility, the N-terminal half of RCK1 undergoes a substantial reorientation relative to the rest of the BK channel gating ring upon Ca2+ binding (Yuan et al., 2012). However, determining whether a CTD/PDG interface exists and contributes to coupling may have to wait until a complete BK channel structure is available.The BK channel CTD contains two high affinity Ca2+-binding sites (one in each RCK domain) that have been identified by site-directed mutagenesis (Bao et al., 2004; Zhang et al., 2010). By using mutations to eliminate each site individually and carefully measuring the effects of Ca2+ on Po at −80 mV (similar to Fig. 2 B), Sweet and Cox (2008) determined that the contribution to Ca2+-sensor/gate coupling of the RCK1 site (3.74 kCal mol−1) was slightly greater than that of the RCK2 Ca2+ bowl site (3.04 kCal mol−1), but the RCK2 site has a higher affinity for Ca2+. In addition, the summed contribution of the individual sites exceeded their combined effect in the WT channel (; Fig. 2 B), consistent with negative cooperativity between sites in the same subunit (Sweet and Cox, 2008). In contrast, another group performing similar experiments at +50 mV concluded that positive cooperativity exists between the two binding sites (Qian et al., 2006). In the latter case, positive cooperativity could potentially be accounted for if both Ca2+ sites were coupled to the voltage sensor because the voltage sensor is not held in a resting state at +50 mV. However Sweet and Cox (2008) concluded that the RCK1 site is solely responsible for Ca2+-sensor/voltage-sensor coupling. A recent study combining Ca2+ site mutations and voltage clamp fluorometry to monitor the effects of Ca2+ on steady-state voltage-sensor activation and Po reached conclusions similar to that of Sweet and Cox (2008) regarding intrasubunit cooperativity and Ca2+-sensor/voltage-sensor coupling (Savalli et al., 2012). However, this study also suggested that Ca2+ site mutations may have effects other than elimination of Ca2+ binding. This caveat, together with the fact that none of the studies mentioned measured Ca2+-sensor/voltage-sensor coupling directly, suggests that the extent to which the individual Ca2+-binding sites are coupled to the voltage sensor or to each other, as well as the molecular mechanisms mediating such interactions, is still open to question.Although the CTD contains two high affinity Ca2+-binding sites, only the Ca2+ bowl is occupied in the Ca2+-bound gating-ring structure (Yuan et al., 2012), raising questions of to what extent this structure resembles the Ca2+-saturated conformation in the intact channel. In principal, the two Ca2+ sites could have independent effects on activation by stabilizing a single Ca2+-bound open conformation. However, mutations in the AC region of RCK1 selectively alter the Ca2+ sensitivity of the RCK1 site (Yang et al., 2010), suggesting that some conformational changes may be coupled to RCK1 occupancy alone. This, together with the fact that the gating ring of the intact channel is expected to contact the VSD, suggests that significant differences in structure could exist between the crystal structure and intact Ca2+-saturated gating ring. A related question is to what extent gating ring expansion is determined by channel opening (i.e., RCK1-S6 linker tension) versus Ca2+ binding. Because gating ring expansion is observed upon Ca2+ binding in isolated gating rings, it seems reasonable to suppose that expansion and channel opening might not occur simultaneously. If the gating ring could expand into a high-Ca2+-affinity conformation while the channel is closed, it could have important implications for gating models and for the possible state dependence of CTD/VSD coupling. However, there is as yet no evidence supporting this possibility. Indeed Sweet and Cox (2008) concluded that their results were best fit by assuming gating-ring expansion is tightly coupled to channel opening, consistent with the HA model.A final question relates to conformational events that occur in Ca2+-binding sites during channel opening. As in any allosteric model of ligand-dependent gating, the HA model predicts that Ca2+-binding sites must have a higher affinity for Ca2+ in the open than the closed conformation. Therefore, understanding how Ca2+ coordination changes upon channel opening is fundamental to the mechanism of Ca2+-sensor/gate coupling. In principal, the contribution of individual Ca2+-coordinating residues to state-dependent binding can be evaluated by determining the effect on coupling energy of mutating such sites (Purohit et al., 2012). Previous studies have generally identified likely Ca2+-coordination sites based on a reduced sensitivity of mutated channels to Ca2+ in the physiological range (≤100 µM). However, experiments at higher [Ca2+] may be required to saturate mutated sites and determine if Ca2+-sensor/gate coupling is altered.Summary
The BK channel is an important example of a voltage- and ligand-gated channel whose function depends on conformational coupling between multiple domains. Many questions remain about the molecular basis of domain–domain coupling, but this channel represents a favorable system for studying such processes owing to its unique functional properties and methods that have allowed the energetics of coupling to be studied in detail. Combining these methods with emerging structural information about domain/domain interfaces and conformational changes in the channel should provide further insight into coupling mechanisms that are important in BK channels and in other voltage-gated or ligand-gated channels. BK channels also constitute a powerful system for understanding the interplay between ligand- and voltage-dependent gating. Defining the interactions that mediate coupling between voltage and Ca2+ sensors in this channel should provide unique insight into processes that may be relevant to other multimodal channels such as HCN or TRP.This Perspectives series includes articles by Andersen, Colquhoun and Lape, and Chowdury and Chanda. 相似文献8.
A number of voltage-activated and Ca2+ activated K+ currents are known to coexist and play a major role in a wide variety of cellular processes including neuromuscular phenomena. Separation of these currents is important for analyzing their individual functional roles and for understanding whether or not they are mediated by entirely different channels. In Drosophila, we have now been able to manipulate four different K+ currents, individually and in combination with one another, by a combined use of mutations and pharmacological agents. This allows analysis of the physiological and molecular properties of different K+ channels and of the role of individual currents in membrane excitability. 相似文献
9.
Chun Chiang 《Bulletin of mathematical biology》1985,47(2):317-320
An equation with five exponential terms is shown to fit the depolarized potassium current with different initial conditions. This equation has a molecular basis and was derived from the assumption that the potassium ions are transported under the combined forces via Newton's second law. It points out that the initial phase of the potassium current may take a different form and the usual concept that potassium conductance has a time delay may be misleading. 相似文献
10.
"Whole-cell" patch recordings using nystatin permeabilization were made from single human platelets during application of agonists from a "puffer" pipette. In platelets clamped near the resting potential and bathed in Na+ saline, 40 microM ADP activated a transient inward current within tens of milliseconds. At -73 mV the current lasted between 0.1 and 1 s and had a peak of between 13 and 31 pA in different cells. Ion substitution experiments indicated that the channel is permeable to Na+,K+, and Ba2+ and presumably also to Ca2+, but is not permeable to Cl-. The single channel conductance was 15 pS (near the resting potential) in nominally Ca(2+)-free saline and 11 picosiemens in BaCl2 saline. Thrombin, at 1 unit/ml, did not elicit detectable currents during a 3-s application in platelets bathed in 1 mM Ca2+, Na+ saline. Under the same conditions, in fura-2-loaded cells, thrombin-evoked Ca2+ entry (monitored by Mn2+ quench) was detectable after a delay of 1.4 s. This suggests that early thrombin-evoked Ca2+ entry occurs via small conductance channels, below the resolution of the patch clamp technique, or by an electroneutral pathway. The ADP-evoked channel has the requisite speed of activation to account for the rapid Ca2+ influx observed during stopped-flow studies of agonist-evoked changes in [Ca2+]i. 相似文献
11.
Properties of calcium and potassium currents of clonal adrenocortical cells 总被引:1,自引:2,他引:1
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The ionic currents of clonal Y-1 adrenocortical cells were studied using the whole-cell variant of the patch-clamp technique. These cells had two major current components: a large outward current carried by K ions, and a small inward Ca current. The Ca current depended on the activity of two populations of Ca channels, slow (SD) and fast (FD) deactivating, that could be separated by their different closing time constants (at -80 mV, SD, 3.8 ms, and FD, 0.13 ms). These two kinds of channels also differed in (a) activation threshold (SD, approximately -50 mV; FD, approximately -20 mV), (b) half-maximal activation (SD, between -15 and -10 mV; FD between +10 and +15 mV), and (c) inactivation time course (SD, fast; FD, slow). The total amplitude of the Ca current and the proportion of SD and FD channels varied from cell to cell. The amplitude of the K current was strongly dependent on the internal [Ca2+] and was almost abolished when internal [Ca2+] was less than 0.001 microM. The K current appeared to be independent, or only slightly dependent, of Ca influx. With an internal [Ca2+] of 0.1 microM, the activation threshold was -20 mV, and at +40 mV the half-time of activation was 9 ms. With 73 mM external K the closing time constant at -70 mV was approximately 3 ms. The outward current was also modulated by internal pH and Mg. At a constant pCa gamma a decrease of pH reduced the current amplitude, whereas the activation kinetics were not much altered. Removal of internal Mg produced a drastic decrease in the amplitude of the Ca-activated K current. It was also found that with internal [Ca2+] over 0.1 microM the K current underwent a time-dependent transformation characterized by a large increase in amplitude and in activation kinetics. 相似文献
12.
13.
During resorption of mineralized tissues, osteoclasts are exposed to marked changes in the concentration of extracellular Ca2+ and H+. We examined the effects of these cations on two types of K+ currents previously described in these cells. Whole-cell patch clamp recordings of membrane currents were made from osteoclasts freshly isolated from neonatal rats. In control saline (1 mm Ca2+, pH 7.4), the voltage-gated, outwardly rectifying K+ current activates at approximately 45 mV and the conductance is half-maximally activated at –29 mV (V
0.5). Increasing [Ca2+]out rapidly and reversibly shifted the current-voltage (I–V) relation to more positive potentials. Current at –29 mV decreased to 28 and 9% of control current at 5 and 10 mm [Ca2+]out, respectively. This effect of elevating [Ca2+]out was due to a positive shift of the K+ channel voltage activation range. Zn2+ or Ni2+ (5 to 500 m) also shifted the I–V relation to more positive potentials and had additional effects consistent with blockade of the K+ channel. Based on the extent to which these divalent cations affected the voltage activation range of the outwardly rectifying K+ current, the potency sequence was Zn2+ > Ni2+ > Ca2+. Lowering or raising extracellular pH also caused shifts of the voltage activation range to more positive or negative potentials, respectively. In contrast to their effects on the outwardly rectifying K+ current, changes in the concentration of extracellular H+ or Ca2+ did not shift the voltage activation range of the inwardly rectifying K+ current. These findings are consistent with Ca2+ and other cations affecting voltage-dependent gating of the osteoclast outwardly rectifying K+ channel through changes in surface charge.This work was supported by The Arthritis Society and the Medical Research Council of Canada. S.M.S. is supported by a Scientist Award and S.J.D. by a Development Grant from the Medical Research Council. 相似文献
14.
Functional hyperemia is an important metabolic autoregulation mechanism by which increased neuronal activity is matched by a rapid and regional increase in blood supply. This mechanism is facilitated by a process known as "neurovascular coupling"--the orchestrated communication system involving neurons, astrocytes and arterioles. Important steps in this process are the production of EETs in the astrocyte and the release of potassium, via two potassium channels (BK and KIR), into the perivascular space. We provide a model which successfully accounts for several observations seen in experiment. The model is capable of simulating the approximate 15% arteriolar dilation caused by a 60-s neuronal activation (modelled as a release of potassium and glutamate into the synaptic cleft). This model also successfully emulates the paradoxical experimental finding that vasoconstriction follows vasodilation when the astrocytic calcium concentration (or perivascular potassium concentration) is increased further. We suggest that the interaction of the changing smooth muscle cell membrane potential and the changing potassium-dependent resting potential of the KIR channel are responsible for this effect. Finally, we demonstrate that a well-controlled mechanism of potassium buffering is potentially important for successful neurovascular coupling. 相似文献
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Summary The effect of pressure upon the delayed, K, voltage-clamp currents of giant axons from the squidLoligo vulgaris was studied in axons treated with 300nm TTX to block the early, Na, currents. The effect of TTX remained unaltered by pressure. The major change produced by pressures up to 62 MPa is a slowing down of the rising phase of the K currents by a time scaling factor which depends on pressure according to an apparent activation volume, V, of 31 cm3/mole at 15°C; V increased to about 42 cm3/mole at 5°C.Pressure slightly increased the magnitude, but did not produce any obvious major change in the voltage dependence, of the steady-state K conductance estimated from the current jump at the end of step depolarizations of small amplitude (to membrane potentials,E, 20 mV) and relatively short duration. At higher depolarizations, pressure produced a more substantial increase of the late membrane conductance, associated with an apparent enhancement of a slow component of the K conductance which could not be described within the framework of the Hodgkin-Huxley (HH)n
4 kinetic scheme.The apparent V values that characterize the pressure dependence of the early component of the K conductance are very close to those that describe the effect of pressure on Na activation kinetics, and it is conceivable that they are related to activation volumes involved in the isomerization of the normal K channels. The enhancement of the slow component of membrane conductance by pressure implies either a large increase in the conductance of the ionic channels that are responsible for it or a strong relative hastening of their turn-on kinetics. 相似文献
18.
W D Nightingale R M Pitman 《Comparative biochemistry and physiology. A, Comparative physiology》1989,93(1):85-93
1. Membrane currents have been recorded from the soma of a bifunctional basalar/coxal depressor motoneurone in the metathoracic ganglion of the cockroach (Periplaneta americana) using a two-electrode voltage-clamp technique. 2. This motoneurone cell body is normally inexcitable when studied under current-clamp. Appropriate depolarizing command steps evoke rapid transient outward currents and late outward currents. 3. Late outward currents are dominated by a Ca-dependent component that confers an N-shaped I-V relationship on the neurone. 4. The Ca-dependent outward current is suppressed by Cd2+ (1 mM), Mn2+ (5 mM) or verapamil (50 microM). 5. Externally applied tetraethylammonium ions (TEA+) (25 mM) block the Ca-dependent current, but also appear to suppress a component of the late outward current that is independent of Ca2+. 6. Aminopyridines cause only minor suppression of late outward currents, but shift the peak in the N-shaped I-V relationship to more negative potentials. 7. The reversal potential of tail currents recorded following pre-pulses to +50 mV were dependent upon the pre-pulse duration; increasing the duration from 10 to 50 msec caused a +17 mV shift in tail current reversal potential. 8. A five-fold increase in the K+ concentration of the solution bathing the preparation only produced small and inconsistent changes in the reversal potential of tail currents. 9. Five-fold reduction in external Cl- caused no change. 10. The dependence of tail current reversal potential upon pre-pulse duration and the limited effect of alterations in the composition of the bathing solution are discussed in the context of restricted ion movements near the external surface of the cell membrane. 相似文献
19.
Previous experiments on cholinergic synapses in chick cochlear hair cells have shown that calcium entering through acetylcholine-activated synaptic channels in turn activates calcium-dependent potassium currents, resulting in synaptic inhibition. In voltage-clamp experiments such currents would be expected to increase with depolarization (as the driving force for potassium entry is increased) and then decrease towards zero as the membrane approaches the calcium equilibrium potential (when calcium entry is suppressed). In the hair cells, however, such currents approached zero at about +20 mV, more than 170 mV negative to the calcium equilibrium potential. Another feature of the synapse is its post-junctional morphology: a uniform 20 nm cleft is formed between the postsynaptic membrane and the outermost membrane of an underlying cisterna. Here we present a model in which synaptic activation results in calcium influx into the subsynaptic cleft and thence into the bulk of the cytoplasm. The model suggests that the voltage dependence of the calcium-activated potassium current can be accounted for by only two basic assumptions: (i) entry of calcium through the activated synaptic channels by simple diffusion; and (ii) activation of the potassium channels by the cooperative action of four calcium ions. In addition, the model suggests that during activation the calcium concentration in the restricted subsynaptic space can reach levels adequate to activate the potassium channels, without requiring additional, more complicated, considerations (for example, secondary calcium release from the cisterna). 相似文献
20.
The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K. 相似文献