Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains

Opening and closing of a CFTR Cl(-) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.     

Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and gamma-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Cl- channel

When excised inside-out membrane patches are bathed in symmetrical Cl--rich solutions, the current-voltage (I-V) relationship of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents inwardly rectifies at large positive voltages. To investigate the mechanism of inward rectification, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human and murine CFTR using voltage-ramp and -step protocols. Using a voltage-ramp protocol, the magnitude of human CFTR Cl- current at +100 mV was 74 +/- 2% (n = 10) of that at -100 mV. This rectification of macroscopic CFTR Cl- current was reproduced in full by ensemble currents generated by averaging single-channel currents elicited by an identical voltage-ramp protocol. However, using a voltage-step protocol the single-channel current amplitude (i) of human CFTR at +100 mV was 88 +/- 2% (n = 10) of that at -100 mV. Based on these data, we hypothesized that voltage might alter the gating behavior of human CFTR. Using linear three-state kinetic schemes, we demonstrated that voltage has marked effects on channel gating. Membrane depolarization decreased both the duration of bursts and the interburst interval, but increased the duration of gaps within bursts. However, because the voltage dependencies of the different rate constants were in opposite directions, voltage was without large effect on the open probability (Po) of human CFTR. In contrast, the Po of murine CFTR was decreased markedly at positive voltages, suggesting that the rectification of murine CFTR is stronger than that of human CFTR. We conclude that inward rectification of CFTR is caused by a reduction in i and changes in gating kinetics. We suggest that inward rectification is an intrinsic property of the CFTR Cl- channel and not the result of pore block.     

CFTR gating I: Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (DeltaR-CFTR)

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed DeltaR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the DeltaR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal DeltaR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of DeltaR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and DeltaR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the DeltaR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.     

Effects of ultraviolet modification on the gating energetics of cyclic nucleotide-gated channels

Middendorf et al. (Middendorf, T.R., R.W. Aldrich, and D.A. Baylor. 2000. J. Gen. Physiol. 116:227-252) showed that ultraviolet light decreases the current through cloned cyclic nucleotide-gated channels from bovine retina activated by high concentrations of cGMP. Here we probe the mechanism of the current reduction. The channels' open probability before irradiation, P(o)(0), determined the sign of the change in current amplitude that occurred upon irradiation. UV always decreased the current through channels with high initial open probabilities [P(o)(0) > 0.3]. Manipulations that promoted channel opening antagonized the current reduction by UV. In contrast, UV always increased the current through channels with low initial open probabilities [P(o)(0) < or = 0.02], and the magnitude of the current increase varied inversely with P(o)(0). The dual effects of UV on channel currents and the correlation of both effects with P(o)(0) suggest that the channels contain two distinct classes of UV target residues whose photochemical modification exerts opposing effects on channel gating. We present a simple model based on this idea that accounts quantitatively for the UV effects on the currents and provides estimates for the photochemical quantum yields and free energy costs of modifying the UV targets. Simulations indicate that UV modification may be used to produce and quantify large changes in channel gating energetics in regimes where the associated changes in open probability are not measurable by existing techniques.     

The CLC ‘chloride channel’ family: revelations from prokaryotes (Review)

Members of the CLC ‘chloride channel’ family play vital roles in a wide variety of physiological settings. Research on prokaryotic CLC homologues provided long-anticipated high-resolution structures as well as the unexpected discovery that some CLCs are not chloride channels, but rather are proton-chloride antiporters. Hence, CLCs encompass two functional classes of transport proteins once thought to be fundamentally different from one another. In this review, we discuss the structural features and molecular mechanisms of CLC channels and antiporters. We focus on ClC-0, the most thoroughly studied CLC channel, and ClC-ec1, the prokaryotic antiporter of known structure. We highlight some striking similarities between these CLCs and discuss compelling questions that remain to be addressed. Prokaryotic CLCs will undoubtedly continue to shed light upon this understudied family of proteins.     

Voltage sensitivity and gating charge in Shaker and Shab family potassium channels.

The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, approximately 13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge-voltage relationships. We find that Shab has a relatively small gating charge, approximately 7.5 e(o). Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.     

Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (tauc) decreased with increasing [ATP] to a minimum value of approximately 0.43 s at [ATP] >1.00 mM. The open time constant (tauo) increased with increasing [ATP] with a minimal tauo of approximately 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of approximately 250 ms is dominant at low ATP concentrations (10 microM) and a much longer open state with a time constant of approximately 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent.     

Fast and slow voltage sensor movements in HERG potassium channels

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH₂-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals.     

囊性纤维化跨膜电导调节体:ATP结合和水解门控Cl-通道

囊性纤维化跨膜电导调节体（cystic fibrosis transmembrane conductance regulator,CFTR）是一种Cl^-通道,属于ATP结合（ATP-binding cassette,ABC）转运体超家族。CFTR功能缺陷是高加索人种中普遍存在的致死性常染色体隐性遗传疾病囊性纤维化（cystic fibrosis,CF）发生的主要原因。这种疾病患者各组织上皮细胞内Cl^-转运失调。目前,与CF相关的不同突变超过1400种。CFTR调节（regulatory,R）域负责调控,核苷酸结合域（nucleotide-binding domains,NBDs）NBD1和NBD2负责ATP结合和水解门控。近期研究发现CFFR的NBDs与其它ABC蛋白一样可以二聚化。二聚化过程中,NBD1和NBD2首-尾相连,一个NBD上的WalkerA和B模块与另一个NBD提供的标签序列（signature sequence）形成ATP结合袋（ATP-binding pockets,ABPs）ABP1和ABP2。ABPs中与ATP结合相关的氨基酸突变实验揭示,ABP1和ABP2在CFTR的ATP依赖门控中发挥不同作用。ABP2由NBD2上的WalkA和B模块与NBD1提供的标签序列形成,它与ATP结合催化通道开放,而ABP1单独与ATP结合不能促进通道开放,只能稳定通道构象。有一些CFrR突变相关疾病的特征就是门控失调,进一步深入研究CFTR的NBD1和NBD2如何通过相互作用而达到通道门控,将为药理学研究提供更多所需的机制信息,有利于为CF治疗的药物设计铺平道路。     

Electrostatic control and chloride regulation of the fast gating of ClC-0 chloride channels

The opening and closing of chloride (Cl-) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as "fast gating" is regulated by both external and internal Cl- ions. Although the external Cl- effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl- regulation remains to be characterized. In this study, we examine the internal Cl- effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising [Cl-]i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of >1 M, a value very different from the one observed in the external Cl- regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties-the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of [Cl-]i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl- on the fast gating may also be due to the competition of Cl- with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl-. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of [Cl-]i and charges in the inner pore region. The very different nature of external and internal Cl- regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.     

Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl− currents by the chemical solvent tetrahydrofuran

The chemical solvent tetrahydrofuran (THF) increases short-circuit current (I_sc) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases I_sc, we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased I_sc in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (K_D) of 134?mM. This THF-induced increase in I_sc was enhanced by forskolin (10 µM), inhibited by the PKA inhibitor H-89 (10 µM) and the thiazolidinone CFTR_inh-172 (10 µM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100?mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase I_sc in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100?mM) potentiated CFTR Cl^? channels open in the presence of ATP (1?mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75?nM), THF (100?mM) did not potentiate channel activity. Similar results were obtained with the ?R-S660A-CFTR Cl^? channel that is not regulated by PKA-dependent phosphorylation and using 2′deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.     

External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba(2+) binding kinetics and the concentration and voltage dependence of Ba(2+) steady-state block. Our results indicate that extracellular Ba(2+) exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba(2+) site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba(2+) site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba(2+) on channel gating in low external K(+) solutions. Ba(2+) binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K(+) attenuates Ba(2+) inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K(+) channels, KCNQ1 channels display significant structural and functional uniqueness.     

Conversion of the 2 Cl−/1 H+ antiporter ClC‐5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl⁻/1 H⁺. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO₃⁻/H⁺ antiporter atClCa, led to coupled NO₃⁻/H⁺ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO₃⁻/H⁺ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.     

Direct Interaction between the Voltage Sensors Produces Cooperative Sustained Deactivation in Voltage-gated H+ Channel Dimers

The voltage-gated H⁺ channel (Hv) is a voltage sensor domain-like protein consisting of four transmembrane segments (S1–S4). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively. Here we show that the two voltage sensor S4 helices within the dimer directly cooperate via a π-stacking interaction between Trp residues at the middle of each segment. Scanning mutagenesis showed that Trp situated around the original position provides the slow gating kinetics characteristic of the dimer''s cooperativity. Analyses of the Trp mutation on the dimeric and monomeric channel backgrounds and analyses with tandem channel constructs suggested that the two Trp residues within the dimer are functionally coupled during Hv deactivation but are less so during activation. Molecular dynamics simulation also showed direct π-stacking of the two Trp residues. These results provide new insight into the cooperative function of voltage-gated channels, where adjacent voltage sensor helices make direct physical contact and work as a single unit according to the gating process.     

The pore structure and gating mechanism of K2P channels

Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.     

Integrated allosteric model of voltage gating of HCN channels

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.     

Coupling between voltage sensor activation,Ca2+ binding and channel opening in large conductance (BK) potassium channels

To determine how intracellular Ca(2+) and membrane voltage regulate the gating of large conductance Ca(2+)-activated K(+) (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca(2+) over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305-336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). In 0 Ca(2+), the steady-state gating charge-voltage (Q(SS)-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (G(K)-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 microM Ca(2+). This change reflects a differential effect of Ca(2+) on voltage sensor activation and channel opening. Ca(2+) has only a small effect on the fast component of ON gating current, indicating that Ca(2+) binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than -80 mV) increases more than 1,000-fold in 70 microM Ca(2+), demonstrating that Ca(2+) increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca(2+) binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca(2+) sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca(2+) sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic I(K) kinetics indicate that Ca(2+) and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.