Adenosine Triphosphate Activates a Noninactivating K+ Current in Adrenal Cortical Cells through Nonhydrolytic Binding

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K⁺ current (I_AC) that is inhibited by adrenocorticotropic hormone and angiotensin II at subnanomolar concentrations. Since I_AC appears to set the membrane potential of AZF cells, these channels may function critically in coupling peptide receptors to membrane depolarization, Ca²⁺ entry, and cortisol secretion. I_AC channel activity may be tightly linked to the metabolic state of the cell. In whole cell patch clamp recordings, MgATP applied intracellularly through the patch electrode at concentrations above 1 mM dramatically enhanced the expression of I_AC K⁺ current. The maximum I_AC current density varied from a low of 8.45 ± 2.74 pA/pF (n = 17) to a high of 109.2 ± 26.3 pA/pF (n = 6) at pipette MgATP concentrations of 0.1 and 10 mM, respectively. In the presence of 5 mM MgATP, I_AC K⁺ channels were tonically active over a wide range of membrane potentials, and voltage-dependent open probability increased by only ∼30% between −40 and +40 mV. ATP (5 mM) in the absence of Mg²⁺ and the nonhydrolyzable ATP analog AMP-PNP (5 mM) were also effective at enhancing the expression of I_AC, from a control value of 3.7 ± 0.1 pA/pF (n = 3) to maximum values of 48.5 ± 9.8 pA/pF (n = 11) and 67.3 ± 23.2 pA/pF (n = 6), respectively. At the single channel level, the unitary I_AC current amplitude did not vary with the ATP concentration or substitution with AMP-PNP. In addition to ATP and AMP-PNP, a number of other nucleotides including GTP, UTP, GDP, and UDP all increased the outwardly rectifying I_AC current with an apparent order of effectiveness: MgATP > ATP = AMP-PNP > GTP = UTP > ADP >> GDP > AMP and ATP-γ-S. Although ATP, GTP, and UTP all enhanced I_AC amplitude with similar effectiveness, inhibition of I_AC by ACTH (200 pM) occurred only in the presence of ATP. As little as 50 μM MgATP restored complete inhibition of I_AC, which had been activated by 5 mM UTP. Although the opening of I_AC channels may require only ATP binding, its inhibition by ACTH appears to involve a mechanism other than hydrolysis of this nucleotide. These findings describe a novel form of K⁺ channel modulation by which I_AC channels are activated through the nonhydrolytic binding of ATP. Because they are activated rather than inhibited by ATP binding, I_AC K⁺ channels may represent a distinctive new variety of K⁺ channel. The combined features of I_AC channels that allow it to sense and respond to changing ATP levels and to set the resting potential of AZF cells, suggest a mechanism where membrane potential, Ca²⁺ entry, and cortisol secretion could be tightly coupled to the metabolic state of the cell through the activity of I_AC K⁺ channels.     

Augmented Insulinotropic Action of Arachidonic Acid through the Lipoxygenase Pathway in the Obese Zucker Rat

Objective: The metabolism of arachidonic acid (AA) has been shown to be altered in severe insulin resistance that is present in obese (fa/fa) Zucker rats. We examined the effects and mechanism of action of AA on basal and glucose‐stimulated insulin secretion in pancreatic islets isolated from obese (fa/fa) Zucker rats and their homozygous lean (Fa/Fa) littermates. Research Methods and Procedures: Islets were isolated from 10‐ to 12‐week‐old rats and incubated for 45 minutes in glucose concentrations ranging from 3.3 to 16.7 mM with or without inhibitors of the cyclooxygenase or lipoxygenase pathways. Medium insulin concentrations were measured by radioimmunoassay, and islet production of the 12‐lipoxygenase metabolite, 12‐hydroxyeicosatetraenoic acid (12‐HETE), was measured by enzyme immunoassay. Results: In islets from lean animals, AA stimulated insulin secretion at submaximally stimulatory glucose levels (< 11.1 mM) but not at 16.7 mM glucose. In contrast, in islets derived from obese rats, AA potentiated insulin secretion at all glucose concentrations. AA‐induced insulin secretion was augmented in islets from obese compared with lean rats at high concentrations of AA in the presence of 3.3 mM glucose. Furthermore, the inhibitor of 12‐lipoxygenase, esculetin (0.5 μM), inhibited AA‐stimulated insulin secretion in islets from obese but not lean rats. Finally, the islet production of the 12‐HETE was markedly enhanced in islets from obese rats, both in response to 16.7 mM glucose and to AA. Discussion: The insulin secretory response to AA is augmented in islets from obese Zucker rats by a mechanism related to enhanced activity of the 12‐lipoxygenase pathway. Therefore, augmented action of AA may be a mechanism underlying the adaptation of insulin secretion to the increased demand caused by insulin resistance in these animals.     

The Binding of Donepezil with External Mouth of K+-Channels of Molluscan Neurons

Earlier, we have shown a strong inhibitory effect of donepezil on K⁺-current of molluscan neurons (Solntseva et al., Comp Biochem Physiol 144, 319–326, 2007). In the present work, a possible interaction of donepezil with the external mouth of the channel was examined using, as a tool, tetraethylammonium (TEA), a classical antagonist of potassium channels. Experiments were conducted in isolated neurons of snail Helix aspersa using the two-microelectrode voltage-clamp technique. A high-threshold slow-inactivating K⁺-current involving Ca²⁺-dependent (I _C) and Ca²⁺-independent (I _K) components was recorded. The I _C was estimated at 30 mV, and I _K at 100 mV. The IC₅₀ values for blocking effect of donepezil on I _C varied from 5.0 to 8.9 μM in different cells. Corresponding values for I _K varied from 4.9 to 9.9 μM. The IC₅₀ values for blocking effect of TEA on I _C lied in the range of 200 to 910 μM, and on I _K lied in the range of 100 to 990 μM. The comparison of the effects of donepezil and TEA on the same cells revealed significant correlation between IC₅₀ values of these effects. The value of Spearman coefficient of correlation (r) was 0.77 for I _C (P < 0.05), and 0.82 for I _K (P < 0.05). In the presence of TEA, the effect of donepezil, both on I _C and I _K, appears significantly weaker than in control solution. Dose–response curves of donepezil effect both on I _C and I _K were shifted right along horizontal axis when donepezil was applied in combination with TEA. Results suggest that TEA interferes with donepezil and precludes the occupation by donepezil of its own site. We suppose that the site for donepezil is situated near the TEA site with possible overlap.     

Extracellular pH Modulates the Voltage-dependent Ca2+ Current and Low Threshold K+ Current in Hair Cells

Extracellular protons have been shown to modulate voltage-activated ionic channels. It has been proposed that synaptic modulation by exocytosed vesicular protons would be a characteristic feature of ribbon-type synapses. Type-I hair cells have a calyceal afferent junction with a diffusionally restricted synaptic cleft. These led us to study the action of extracellular pH changes on the voltage-activated Ca²⁺ and K⁺ currents evaluated using a whole-cell patch clamp in isolated cells. The amplitude of the Ca²⁺ and the K⁺ current were reduced by extracellular acidification, but without significant changes with extracellular alkalization. A shift in the voltage dependence to a more positive membrane potential was achieved at pH < 6.8. Our results shows that the presynaptic K⁺ and Ca²⁺ currents are modulated by protons, indicating that protons released along with an afferent neurotransmitter would participate as a feedback mechanism in type-I hair cells. Special issue article in honor of Dr. Ricardo Tapia.     

Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

Prolonged depolarization induces a slow inactivation process in some K⁺ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K⁺ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K⁺ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 microM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 +/- 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 +/- 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2-5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an "IQ-like" motif present in the carboxy terminus of KCNQ2-5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic "gene gun." Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.     

Lipoxygenase Pathway of Arachidonic Acid Metabolism in Growth Control of Tumor Cells of Different Type

The influence of inhibitors of different lipoxygenases (LOX) on the growth of human tumor cells with different profiles of synthesized eicosanoids was studied. The studied LOX inhibitors had virtually no influence on the growth of A549 cells actively synthesizing cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA). The inhibitor of 12-LOX, baicalein, significantly inhibited proliferation in cultures of A431 epidermoid carcinoma cells with a characteristic domination of the major lipoxygenase metabolite of AA, 12-hydroxyeicosatetraenoic acid (12-HETE), in the profile of synthesized eicosanoids and reduced to 70% the incorporation of [3H]thymidine into DNA. Treatment of these cultures with 12-HETE virtually restored the growth potential of the tumor cells. The findings suggest that the lipoxygenase metabolite of AA, 12-HETE, is a growth-limiting factor for tumor cells of definite type.     

Construction of a cyclic nucleotide-gated KcsA K+ channel

The ability of an ion channel to open in response to a defined stimulus is central to its function. In ligand-gated channels, pore opening is conferred through transduction of a conformational change in a gating domain to the helices of the pore. Here, we present the construction of a designed cyclic nucleotide-gated (CNG) channel, named KcsA-CNG, by addition of a prokaryotic cyclic nucleotide-binding domain to a KcsA-derived K+ channel. This channel is functional in lipid bilayers at physiological pH and has the combined properties of both of its parent-derived components. It conducts K+ and is blocked by the K+ channel inhibitors Na+ and agitoxin-2. Channel open times are increased by about two orders of magnitude compared to wild-type KcsA. The average number of open channels increases by approximately 50% upon addition of cAMP. Although the absolute open probabilities are somewhat variable from one channel to the next, the property of cyclic nucleotide sensitivity is very reproducible. An apparent Kd value of approximately 90 nM was estimated. The successful construction of a cyclic nucleotide-gated KcsA K+ channel suggests that it should be possible to produce channels that will respond to novel ligands.     

Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

Summary The patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K₂ malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at –100 mV, in 100mm symmetrical KCl was about 160 S/cm². The reversal potentials (E _rev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K⁺ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na⁺ too can flow through the channels. NeitherE _rev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K⁺ as well as to Na⁺. Anions such as Cl^– or malate seem to contribute little to the channel current.     

The enantiomer pair of 24S- and 24R-hydroxycholesterol differentially alter activity of large-conductance Ca2+-dependent K+ (slo1 BK) channel

24S-hydroxycholesterol (HC) is most abundant oxysterols in the brain, passes through blood brain barrier, and is therefore regarded as an intermediary for brain cholesterol elimination. We reported that large-conductance Ca²⁺- and voltage-activated K⁺ (slo1 BK) channels are suppressed by this oxysterol, which is presumably intercalated into cell membrane to access the outer surface of the channel. Such an outer approach would make it difficult to interact with the inner, ion-conducting part of the channel. The present findings showed that 24R-HC, the racemic counterpart of 24S-HC, also suppressed slo1 BK channel but in a different voltage-dependent manner. There was a difference between the effects of the two enantiomers on activation kinetics but not on deactivation kinetics. It is suggested that the chirality contributes to the efficacy of channel blockers that act from outer lipophilic parts of channels, as with those which act on the inner, ion-permeable surface.     

Heteromeric K+ channels in plants

Voltage-gated potassium channels of plants are multimeric proteins built of four α-subunits. In the model plant Arabidopsis thaliana , nine genes coding for K⁺ channel α-subunits have been identified. When co-expressed in heterologous expression systems, most of them display the ability to form heteromeric K⁺ channels. Till now it was not clear whether plants use this potential of heteromerization to increase the functional diversity of potassium channels. Here, we designed an experimental approach employing different transgenic plant lines that allowed us to prove the existence of heteromeric K⁺ channels in plants. The chosen strategy might also be useful for investigating the activity and function of other multimeric channel proteins like, for instance, cyclic-nucleotide gated channels, tandem-pore K⁺ channels and glutamate receptor channels.     

Voltage-dependent K channels in protoplasts of trap-lobe cells ofDionaea muscipula

Summary The outward rectification of the K⁺ current in mesophyll cell protoplasts from trap-lobes ofDionaea muscipula was studied with the patch-clamp technique. The rectification had instantaneous and time-dependent components. Changes in [K⁺] _i strongly affected the conductance voltage relation of the plasma membrane while changes in [K⁺] _o had little effect on the relation. Thus, the outward rectification depends on the membrane voltage and the concentration of intracellular K⁺. Corresponding single-channel activities were observed both in the intact membrane (cell-attached recording) and in excised patches. The single-channel conductance was about 3.3 pS with symmetrical solutions containing 30mm K⁺.     

Permeance of Cs+ and Rb+ through the inwardly rectifying K+ channel in guinea pig ventricular myocytes

Summary Inward currents carried by external Cs, Rb, NH₄ and K through theI _K1 channel were studied using a whole-cell voltage clamp technique. Cs, NH₄, and Rb currents could be recorded negative to –40 mV following depolarizing prepulses (0 mV and 200–1000 msec in duration). The current activation displayed an instantaneous component followed by a monoexponential increase () to a peak amplitude. Subsequent inactivation was fit by a single exponential, _i. With hyperpolarization, and _i decreasede-fold per 36 and 25 mV, respectively. In Ca-free external solutions (pipette [Mg]0.3mm), inactivation was absent, consistent with the hypothesis that inactivation represents time- and voltage-dependent block of Cs, NH₄, and Rb currents by external Ca. The inactivation and degree of steady-state block was greatest when Cs was the charge carrier, followed by NH₄, and then Rb. K currents, however, did not inactivate in the presence of Ca. Na and Li did not carry any significant current within the resolution of our recordings. Comparison ofpeak inward current ratios (I _x/I_K) as an index of permeability revealed a higher permeance of Cs (0.15), NH₄ (0.30), and Rb (0.51) relative to K (1.0) than that obtained by comparing thesteady-state current ratios (CsNH₄RbK0.010.060.211.0). At any given potential, was smaller the more permeant the cation. In the absence of depolarizing prepulses, the amplitude of was reduced. Divalent-free solutions did not significantly affect activatio in the presence of 0.3mm pipette [Mg]. When pipette [Mg] was buffered to 50 m, however, removal of external Ca and Mg lead to a four- to fivefold increase in Cs currents and loss of both time-dependent activation and inactivation (reversible upon repletion of external Ca).These results suggest that (i) permeability ratios forI _K1 should account for differences in the degree to which monovalent currents are blocked by extracellular Ca and (ii) extracellular or intracellular divalent cations contribute to the slow phase of activation which may represent either (a) the actual rate of Mg or Ca extrusion from the channel into the cell, a process which may be enhanced by repulsive interaction with the incoming permeant monovalent cation or (b) an intrinsic gating process that is strongly modulated by the permeant monovalent ion and divalent cations.     

Neuritin Activates Insulin Receptor Pathway to Up-regulate Kv4.2-mediated Transient Outward K+ Current in Rat Cerebellar Granule Neurons

Neuritin is a new neurotrophic factor discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin also plays multiple roles in the process of neural development and synaptic plasticity. The receptors for binding neuritin and its downstream signaling effectors, however, remain unclear. Here, we report that neuritin specifically increases the densities of transient outward K⁺ currents (I_A) in rat cerebellar granule neurons (CGNs) in a time- and concentration-dependent manner. Neuritin-induced amplification of I_A is mediated by increased mRNA and protein expression of Kv4.2, the main α-subunit of I_A. Exposure of CGNs to neuritin markedly induces phosphorylation of ERK (pERK), Akt (pAkt), and mammalian target of rapamycin (pmTOR). Neuritin-induced I_A and increased expression of Kv4.2 are attenuated by ERK, Akt, or mTOR inhibitors. Unexpectedly, pharmacological blockade of insulin receptor, but not the insulin-like growth factor 1 receptor, abrogates the effect of neuritin on I_A amplification and Kv4.2 induction. Indeed, neuritin activates downstream signaling effectors of the insulin receptor in CGNs and HeLa. Our data reveal, for the first time, an unanticipated role of the insulin receptor in previously unrecognized neuritin-mediated signaling.     

ATP-regulated K+ channel in mitochondria: Pharmacology and function

Mitochondria from several tissues contain a potassium-specific channel similar to the ATP-regulated K⁺ (K_ATP) channel of the plasma membrane. The mitochondrial channel shares with the plasma membrane K_ATP channel the sensitivity to sulfonylurea derivatives and some other blockers as well as to channel openers of diverse chemical character. In contrast to the plasma membrane channel, which is blocked by free ATP, the mitochondrial K_ATP channel reconstituted into liposomes requires the ATP-Mg complex for inhibition. The mitochondrial K_ATP channel, possibly in a concerted action with other K⁺ permeability pathways, plays an important role in mitochondrial volume control. Its function in the regulation of the components of the protonmotive force is also suggested.     

Single-channel properties and pH sensitivity of two-pore domain K+ channels of the TALK family

The two-pore K2P channel family comprises TASK, TREK, TWIK, TRESK, TALK, and THIK subfamilies, and TALK-1, TALK-2, and TASK-2 are functional members of the TALK subfamily. Here we report for the first time the single-channel properties of TALK-2 and its pHo sensitivity, and compare them to those of TALK-1 and TASK-2. In transfected COS-7 cells, the three TALK K2P channels could be identified easily by their differences in single-channel conductance and gating kinetics. The single-channel conductances of TALK-1, TALK-2, and TASK-2 in symmetrical 150 mM KCl were 21, 33, and 70 pS (-60 mV), respectively. TALK-2 was sensitive mainly to the alkaline range (pH 7-10), whereas TALK-1 and TASK-2 were sensitive to a wider pHo range (6-10). The effect of pH changes was mainly on the opening frequency. Thus, members of the TALK family expressed in native tissues may be identified based on their single-channel kinetics and pHo sensitivity.     

Regulation of K+ flow by a ring of negative charges in the outer pore of BKCa channels. Part II: Neutralization of aspartate 292 reduces long channel openings and gating current slow component

Neutralization of the aspartate near the selectivity filter in the GYGD pore sequence (D292N) of the voltage- and Ca(2+)-activated K+ channel (MaxiK, BKCa) does not prevent conduction like the corresponding mutation in Shaker channel, but profoundly affects major biophysical properties of the channel (Haug, T., D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese. 2004. J. Gen. Physiol. 124:173-184). Upon depolarizations, the D292N mutant elicited mostly gating current, followed by small or no ionic current, at voltages where the wild-type hSlo channel displayed robust ionic current. In fact, while the voltage dependence of the gating current was not significantly affected by the mutation, the overall activation curve was shifted by approximately 20 mV toward more depolarized potentials. Several lines of evidence suggest that the mutation prevents population of certain open states that in the wild type lead to high open probability. The activation curves of WT and D292N can both be fitted to the sum of two Boltzmann distributions with identical slope factors and half activation potentials, just by changing their relative amplitudes. The steeper and more negative component of the activation curve was drastically reduced by the D292N mutation (from 0.65 to 0.30), suggesting that the population of open states that occurs early in the activation pathway is reduced. Furthermore, the slow component of the gating current, which has been suggested to reflect transitions from closed to open states, was greatly reduced in D292N channels. The D292N mutation also affected the limiting open probability: at 0 mV, the limiting open probability dropped from approximately 0.5 for the wild-type channel to 0.06 in D292N (in 1 mM [Ca2+]i). In addition to these effects on gating charge and open probability, as already described in Part I, the D292N mutation introduces a approximately 40% reduction of outward single channel conductance, as well as a strong outward rectification.     

Allosteric Gating of a Large Conductance Ca-activated K+ Channel

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca²⁺. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca²⁺ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca²⁺ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca²⁺ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca²⁺ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.