Nonstationary fluctuation analysis of the delayed rectifier K channel in cardiac Purkinje fibers. Actions of norepinephrine on single-channel current.

We have studied the large increase in macroscopic potassium channel current caused by catecholamines in mammalian cardiac cells. An increase in macroscopic K current could result from either an increase in the single-channel current or by an increase in the number of channels that are open. Therefore, we have measured nonstationary potassium current fluctuations under voltage clamp conditions to determine whether norepinephrine increases the current through this channel. The single-channel current (at a potential of -30 mV in 4 mM external [K]) was estimated to be 3.7 pA and was not altered by concentrations of norepinephrine up to 2 microM. The spectral density of the current fluctuations were fitted well by a sum of 2 Lorentzians with corner frequencies that correspond with the measured time constants for deactivation of the macroscopic K current tails. We conclude that the increase in macroscopic K current caused by norepinephrine in these cells is not the result of an increase in single-channel conductance and therefore must involve an increase in the number of open K channels.     

Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated K(+) channels

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl(-) channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K(+) channel of 123 +/- 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K(+) over Na(+) and Ca(2+). The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 microM), and gadolinium (500 microM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K(+) channel activity is consistent with the hypothesis that stretch-activated K(+) channels could mediate an increase in K(+) conductance during cell swelling.     

Effects of gadolinium on ion channels in the myelinated axon of Xenopus laevis: four sites of action.

The action of gadolinium (Gd3+) on ion currents in myelinated axons of Xenopus laevis was investigated with the voltage clamp technique. The analysis revealed the following effects. (i) The potential-dependent parameters of both Na and K channels were shifted. The shift was equally large for activation, inactivation, and activation time constant curves (+9 mV for 100 microM Gd3+). The effects could be explained by screening of fixed surface charges at a density of -1.2 e nm-2. (ii) The rate of gating for both Na and K channels was reduced more than predicted from the shift. This effect could be quantified as a scaling (by a factor 3 and 5 respectively at 100 microM Gd3+) of the activation time constant curves. (iii) An activation- and inactivation-independent block of both Na and K channels, obeying 1:1 stoichiometry with a Kd value of about 70 microM potential-independent block of leakage current, obeying 1:2 stoichiometry with a Kd value of 600 microM. (iv) The analysis suggests separate binding sites for the effects, comprising high affinity modulatory and blocking sites on the channel proteins and low affinity receptors on phospholipids, associated with the effect on the leakage current.     

Voltage-dependent modulation of Ca channel current in heart cells by Bay K8644

We have investigated the voltage-dependent effects of the dihydropyridine Bay K8644 on Ca channel currents in calf Purkinje fibers and enzymatically dispersed rat ventricular myocytes. Bay K8644 increases the apparent rate of inactivation of these currents, measured during depolarizing voltage pulses, and shifts both channel activation and inactivation in the hyperpolarizing direction. Consequently, currents measured after hyperpolarizing conditioning pulses are larger in the presence of drug compared with control conditions, but are smaller than control if they are measured after positive conditioning pulses. Most of our experimental observations on macroscopic currents can be explained by a single drug-induced change in one rate constant of a simple kinetic model. The rate constant change is consistent with results obtained by others with single channel recordings.     

How ATP inhibits the open K(ATP) channel

ATP-sensitive potassium (K(ATP)) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 microM.     

Localization of the K+ lock-In and the Ba2+ binding sites in a voltage-gated calcium-modulated channel. Implications for survival of K+ permeability.

Using Ba2+ as a probe, we performed a detailed characterization of an external K+ binding site located in the pore of a large conductance Ca2+-activated K+ (BKCa) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba2+ blocks BKCa channels and decreasing external K+ using a K+ chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba2+-blocked events. Average Ba2+ dwell time changes from 10 s at 10 mM external K+ to 100 ms in the limit of very low [K+]. Using a model where external K+ binds to a site hindering the exit of Ba2+ toward the external side (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-568), we calculated a dissociation constant of 2.7 mircoM for K) at this lock-in site. We also found that BK(Ca) channels enter into a long-lasting nonconductive state when the external [K+] is reduced below 4 microM using the crown ether. Channel activity can be recovered by adding K+, Rb+, Cs+, or NH4+ to the external solution. These results suggest that the BK(Ca) channel stability in solutions of very low [K+] is due to K+ binding to a site having a very high affinity. Occupancy of this site by K+ avoids the channel conductance collapse and the exit of Ba2+ toward the external side. External tetraethylammonium also reduced the Ba2+ off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires the presence of external K+. It is explained in terms of a model in which the conduction pore contains Ba2+, K+, and tetraethylammonium simultaneously, with the K+ binding site located internal to the tetraethylammonium site. Altogether, these results and the known potassium channel structure (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) imply that the lock-in site and the Ba2+ sites are the external and internal ion sites of the selectivity filter, respectively.     

The facilitative actions of Bay K 8644 on norepinephrine and KCl-induced contractures of rabbit aortic rings

The effect of the calcium channel agonist BAY K 8644 on the ability of KCl and norepinephrine to induce contractions of rabbit aortic rings has been examined in Krebs-Henseleit buffer containing either 4.0 or 6.8 mM potassium. BAY K 8644 (10(-8) to 10(-6) M) alone induced slowly developing aortic contractures which were 10 (at 4.0 mM potassium) or 20 (at 6.8 mM potassium) percent of the maximum obtainable with norepinephrine. These contractions were not observed in every experiment, but were more likely to occur at 6.8 mM (71% at 10(-6) M BAY K 8644) when compared to 4.0 mM (31% at 10(-6) M BAY K 8644) potassium buffer. BAY K 8644, in either potassium buffer, induced a statistically significant shift to the left in the norepinephrine dose-response curve. The norepinephrine dose-response curve was significantly curvilinear in the presence of 3 X 10(-8) M BAY K 8644 (6.8 mM potassium) and 10(-6) M BAY K 8644 (4.0 mM potassium). Similarly, BAY K 8644 induced sinistral shifts in the KCl dose-response curve with a curvilinear function observed at 3 X 10(-7) M BAY K 8644. These data show that BAY K 8644 is capable of inducing aortic contractures at potassium concentrations significantly lower than previously reported. Furthermore, BAY K 8644 facilitates opening of calcium channels by either potassium or norepinephrine. In contrast to others, our data indicates that BAY K 8644 can affect calcium channels activated by norepinephrine. Finally, our data suggest that the alpha and dihydropyridine receptors are capable of interacting and that occupation of one receptor can affect the action of a compound binding to the other receptor.     

Modulation of potassium channel gating by external divalent cations

We have examined the actions of Zn2+ ions on Shaker K channels. We found that low (100 microM) concentrations of Zn2+ produced a substantial (approximately three-fold) slowing of the kinetics of macroscopic activation and inactivation. Channel deactivation was much less affected. These results were obtained in the presence of 5 mM Mg2+ and 4 mM Ca2+ in the external solution and so are unlikely to be due to modification of membrane surface charges. Furthermore, the action of 100 microM Zn2+ on activation was equivalent to a 70-mV reduction of a negative surface potential whereas the effects on deactivation would require a 15-mV increase in surface potential. External H+ ions reduced the Zn-induced slowing of macroscopic activation with an apparent pK of 7.3. Treatment of Shaker K channels with the amino group reagent, trinitrobenzene sulfonic acid (TNBS), substantially reduced the effects of Zn2+. All these results are qualitatively similar to the actions of Zn2+ on squid K channels, indicating that the binding site may be a common motif in potassium channels. Studies of single Shaker channel properties showed that Zn2+ ions had little or no effect on the open channel current level or on the open channel lifetime. Rather, Zn2+ substantially delayed the time to first channel opening. Thus, K channels appear to contain a site to which divalent cations bind and in so doing act to slow one or more of the rate constants controlling transitions among closed conformational states of the channel.     

Functional distinctions between the mitochondrial ATP-dependent K+ channel (mitoKATP) and its inward rectifier subunit (mitoKIR)

The ATP-sensitive potassium channel from the inner mitochondrial membrane (mitoK(ATP)) is a highly selective conductor of K(+) ions. When isolated in the presence of nonionic detergent and reconstituted in liposomes, mitoK(ATP) is inhibited with high affinity by ATP (K((1/2)) = 20-30 microM). We have suggested that holo-mitoK(ATP) is a heteromultimer consisting of an inwardly rectifying K(+) channel (mitoKIR) and a sulfonylurea receptor (Grover, G. J., and Garlid, K. D. (2000) J. Mol. Cell. Cardiol. 32, 677-695). Here, we show that a 55-kDa protein isolated by ethanol extraction and reconstituted in bilayer lipid membranes and liposomes is the mitoKIR. This protein, which lacks the sulfonylurea receptor subunit, is inhibited with low affinity by ATP, with K(1/2) approximately 550 microM. ATP inhibition of both mitoKIR and holo-mitoK(ATP) is reversed by UDP (K((1/2))1/2 = 10-15 microM). Holo-mitoK(ATP) is and diazoxide, and the opened by cromakalim flux through the open channel is inhibited by glibenclamide and 5-hydroxydecanoate. None of these agents has any effect upon mitoKIR. We have identified two compounds that act specifically on mitoKIR. p-diethylaminoethylbenzoate reverses inhibition of mitoKIR by ATP and ADP at micromolar concentrations and also opens mitoK(ATP) in isolated mitochondria. Tetraphenylphosphonium inhibits K(+) flux through both mitoKIR and mitoK(ATP) with the same apparent affinity. These findings support the hypothesis that the 55-kDa mitoKIR is the channel component of mitoK(ATP).     

The calcium-activated potassium channels of turtle hair cells

A major factor determining the electrical resonant frequency of turtle cochlear hair cells is the time course of the Ca-activated K current (Art, J. J., and R. Fettiplace. 1987. Journal of Physiology. 385:207- 242). We have examined the notion that this time course is dictated by the K channel kinetics by recording single Ca-activated K channels in inside-out patches from isolated cells. A hair cell's resonant frequency was estimated from its known correlation with the dimensions of the hair bundle. All cells possess BK channels with a similar unit conductance of approximately 320 pS but with different mean open times of 0.25-12 ms. The time constant of relaxation of the average single- channel current at -50 mV in 4 microM Ca varied between cells from 0.4 to 13 ms and was correlated with the hair bundle height. The magnitude and voltage dependence of the time constant agree with the expected behavior of the macroscopic K(Ca) current, whose speed may thus be limited by the channel kinetics. All BK channels had similar sensitivities to Ca which produced half-maximal activation for a concentration of approximately 2 microM at +50 mV and 12 microM at -50 mV. We estimate from the voltage dependence of the whole-cell K(Ca) current that the BK channels may be fully activated at -35 mV by a rise in intracellular Ca to 50 microM. BK channels were occasionally observed to switch between slow and fast gating modes which raises the possibility that the range of kinetics of BK channels observed in different hair cells reflects a common channel protein whose kinetics are regulated by an unidentified intracellular factor. Membrane patches also contained 30 pS SK channels which were approximately 5 times more Ca-sensitive than BK channels at -50 mV. The SK channels may underlie the inhibitory synaptic potential produced in hair cells by efferent stimulation.     

Profilin promotes barbed-end actin filament assembly without lowering the critical concentration

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin.4.1.Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta4, both actin and Profilin.Actin (P.A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant ( approximately 10 x 10(6) M(-1) s(-1)) for actin monomer or Profilin.Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 microM thymosin-beta4 concentration range fit a simple monomer sequestering model (1 microM K(D) for Thymosin-beta4.Actin). The corresponding constant for thymosin-beta4.pyrenyl-Actin, however, was significantly higher ( approximately 9-10 microM), suggesting that the fluorophore markedly weakens binding to thymosin-beta4. With solutions of actin (2 microM) and thymosin-beta4 (2 or 4 microM), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 microM). Actin assembly in presence of thymosin-beta4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta4.Actin and ushers actin as a Profilin.Actin complex onto growing barbed filament ends.     

Electrostatic calculations for an ion channel. II. Kinetic behavior of the gramicidin A channel.

A theoretical model of the gramicidin A channel is presented and the kinetic behavior of the model is derived and compared with previous experimental results. The major assumption of the model is that the only interaction between ions in a multiply-occupied channel is electrostatic. The electrostatic calculations indicate in a multiply-occupied channel is electrostatic. The electrostatic calculations indicate that there will be potential wells at each end of the channel and, at high concentrations, that both wells can be occupied. The kinetics are based on two reaction steps: movement of the ion from the bulk solution to the well and movement between the two wells. The kinetics for this reaction rate approach are identical to those based on the Nernst-Planck equation in the limit where the movement between the two wells is rate limiting. The experimental results for sodium and potassium are consistent with a maximum of two ions per channel. To explain the thallium results it is necessary to allow three ions per channel. It is shown that this case is compatible with the electrostatic calculations if the presence of an anion is included. The theoretical kinetics are in reasonable quantitative agreement with the following experimental measurements: single channel conductance of sodium, potassium, and thallium; bi-ionic potential and permeability ratio between sodium-potassium and potassium-thallium; the limiting conductance of potassium and thallium at high applied voltages; current-voltage curves for sodium and potassium at low (but not high) concentrations; and the inhibition of sodium conductance by thallium. The results suggest that the potential well is located close to the channel mouth and that the conductance is partially limited by the rate going from the bulk solution to the well. For thallium, this entrance rate is probably diffusion limited.     

Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms by excitatory amino acids and by BAY K-8644 is dependent upon the assay calcium and potassium concentrations used.

The calcium and potassium ion dependency of the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 microM) potentiated the response to carbachol at 6 mM K+ when Ca2(+)-free, but not when 2.52 mM Ca2+ assay buffer was used. In Ca2(+)-free buffer, verapamil (10 microM) inhibited the response to carbachol at both 6 and 18 mM K+ but higher concentrations (30-300 microM) were needed when 2.52 mM Ca2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [3H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 microM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K+ at 1.3 mM Ca2+, but was without effect on the basal rate at other K+ and Ca2+ concentrations. In the presence of NMDA (100 microM) or quisqualate (100 microM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K+ and Ca2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.     

Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K(+) channel. A molecular probe for the mechanism of ATP-sensitive inhibition.

Anionic phospholipids modulate the activity of inwardly rectifying potassium channels (Fan, Z., and J.C. Makielski. 1997. J. Biol. Chem. 272:5388-5395). The effect of phosphoinositides on adenosine triphosphate (ATP) inhibition of ATP-sensitive potassium channel (K(ATP)) currents was investigated using the inside-out patch clamp technique in cardiac myocytes and in COS-1 cells in which the cardiac isoform of the sulfonylurea receptor, SUR2, was coexpressed with the inwardly rectifying channel Kir6.2. Phosphoinositides (1 mg/ml) increased the open probability of K(ATP) in low [ATP] (1 microM) within 30 s. Phosphoinositides desensitized ATP inhibition with a longer onset period (>3 min), activating channels inhibited by ATP (1 mM). Phosphoinositides treatment for 10 min shifted the half-inhibitory [ATP] (K(i)) from 35 microM to 16 mM. At the single-channel level, increased [ATP] caused a shorter mean open time and a longer mean closed time. Phosphoinositides prolonged the mean open time, shortened the mean closed time, and weakened the [ATP] dependence of these parameters resulting in a higher open probability at any given [ATP]. The apparent rate constants for ATP binding were estimated to be 0.8 and 0.02 mM(-1) ms(-1) before and after 5-min treatment with phosphoinositides, which corresponds to a K(i) of 35 microM and 5.8 mM, respectively. Phosphoinositides failed to desensitize adenosine inhibition of K(ATP). In the presence of SUR2, phosphoinositides attenuated MgATP antagonism of ATP inhibition. Kir6.2DeltaC35, a truncated Kir6.2 that functions without SUR2, also exhibited phosphoinositide desensitization of ATP inhibition. These data suggest that (a) phosphoinositides strongly compete with ATP at a binding site residing on Kir6.2; (b) electrostatic interaction is a characteristic property of this competition; and (c) in conjunction with SUR2, phosphoinositides render additional, complex effects on ATP inhibition. We propose a model of the ATP binding site involving positively charged residues on the COOH-terminus of Kir6.2, with which phosphoinositides interact to desensitize ATP inhibition.     

Fractal analysis of a voltage-dependent potassium channel from cultured mouse hippocampal neurons.

The kinetics of ion channels have been widely modeled as a Markov process. In these models it is assumed that the channel protein has a small number of discrete conformational states and the kinetic rate constants connecting these states are constant. In the alternative fractal model the spontaneous fluctuations of the channel protein at many different time scales are represented by a kinetic rate constant k = At1-D, where A is the kinetic setpoint and D the fractal dimension. Single-channel currents were recorded at 146 mM external K+ from an inwardly rectifying, 120 pS, K+ selective, voltage-sensitive channel in cultured mouse hippocampal neurons. The kinetics of these channels were found to be statistically self-similar at different time scales as predicted by the fractal model. The fractal dimensions were approximately 2 for the closed times and approximately 1 for the open times and did not depend on voltage. For both the open and closed times the logarithm of the kinetic setpoint was found to be proportional to the applied voltage, which indicates that the gating of this channel involves the net inward movement of approximately one negative charge when this channel opens. Thus, the open and closed times and the voltage dependence of the gating of this channel are well described by the fractal model.     

Single Na+ channels activated by veratridine and batrachotoxin

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.     

Depressed transient outward potassium current density in catecholamine-depleted rat ventricular myocytes

The effect of catecholamine depletion (induced by prior treatment with reserpine) was studied in Wistar rat ventricular myocytes using whole cell voltage-clamp methods. Two calcium-independent outward currents, the transient outward potassium current (I(to)) and the sustained outward potassium current (I(sus)), were measured. Reserpine treatment decreased tissue norepinephrine content by 97%. Action potential duration in the isolated perfused heart was significantly increased in reserpine-treated hearts. In isolated ventricular myocytes, I(to) density was decreased by 49% in reserpine-treated rats. This treatment had no effect on I(sus). The I(to) steady-state inactivation-voltage relationship and recovery from inactivation remained unchanged, whereas the conductance-voltage activation curve for reserpine-treated rats was significantly shifted (6.7 mV) toward negative potentials. The incubation of myocytes with 10 microM norepinephrine for 7-10 h restored I(to), an effect that was abolished by the presence of actinomycin D. Norepinephrine (0.5 microM) had no effect on I(to). However, in the presence of both 0.5 microM norepinephrine and neuropeptide Y (0.1 microM), I(to) density was restored to its control value. These results suggest that the sympathetic nervous system is involved in I(to) regulation. Sympathetic norepinephrine depletion decreased the number of functional channels via an effect on the alpha-adrenergic cascade and norepinephrine is able to restore expression of I(to) channels.     

The cGMP-dependent channel of vertebrate rod photoreceptors exists in two forms of different cGMP sensitivity and pharmacological behavior

cGMP cooperatively activates a cation-selective channel in vertebrate photoreceptors probably by direct binding to a membrane receptor (Fesenko, E. E., Kolesnikov, S. S., and Lyubarsky, A.L. (1985) Nature 313, 310-313). We have recently described the existence of a similar channel in photoreceptor disc membranes (Koch, K. W., and Kaupp, U. B. (1985) J. Biol. Chem. 260, 6788-6800). In this paper we report that the cGMP-dependent channel in the disc membrane of outer segments exists in at least two forms of different cGMP sensitivity and pharmacological behavior. One form has a low Km value of activation by cGMP (25 microM), and it is not blocked by l-cis-diltiazem. The other form has a high Km value (170 microM) and is blocked by this drug. Both channel forms are cooperatively activated by cGMP (Hill coefficient n = 2.0-3.5), suggesting that the channel may have a cGMP receptor that can bind at least three, and possibly four, molecules of cGMP. In the presence of cGMP, the open state of both channel forms inactivates to a weakly or nonconductive state (cf. Puckett, K. L., and Goldin, S. M. (1986) Biochemistry 25, 1739-1746). A mixed population of different forms of the cGMP-dependent channel may explain the variable numbers reported for the Michaelis constant Km for the channel activation by cGMP and the cooperativity n.     

Blocking kinetics of Cl- channels in colonic carcinoma cells (HT29) as revealed by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)

The blocking effect of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was investigated on single Cl- channels of the cultured human colon carcinoma cells, HT29. In the absence of NPPB, the open-time histogram yielded two time constants, with 0.9 ms and 33 ms, whereas the closed-time distribution could be fitted by a single exponential with a time constant of 0.7 ms. Addition of NPPB in the range 1-50 microM induced brief closing events of the single-channel current. This resulted in a decrease of the long open-time constant to 2.1 ms and in an increase of the closed-time constant to 1.8 ms at 50 microM NPPB concentration. The short open-time constant did not change at low blocker concentration (1 microM), but could no longer be resolved at higher concentrations. The open-state probability decreased from 0.9 (control conditions) to 0.5 at 50 microM NPPB. The Hill plot yielded a Hill coefficient of about 0.7, compatible with one NPPB molecule inhibiting one channel molecule. The kinetics of channel gating are described by a sequential model with one closed and two open states. Since in the presence of NPPB no additional time constant appeared in the time histograms, we assumed the same kinetic scheme as under control conditions, and hypothesize that NPPB has an influence on rate constants.     

Biophysical properties and metabolic regulation of a TASK-like potassium channel in rat carotid body type 1 cells

The single channel properties of TASK-like oxygen-sensitive potassium channels were studied in rat carotid body type 1 cells. We observed channels with rapid bursting kinetics, active at resting membrane potentials. These channels were highly potassium selective with a slope conductance of 14-16 pS, values similar to those reported for TASK-1. In the absence of extracellular divalent cations, however, single channel conductance increased to 28 pS in a manner similar to that reported for TASK-3. After patch excision, channel activity ran down rapidly. Channel activity in inside-out patches was markedly increased by 2 and 5 mM ATP and by 2 mM ADP but not by 100 microM ADP or 1 mM AMP. In cell-attached patches, both cyanide and 2,4-dinitrophenol strongly inhibited channel activity. We conclude that 1) whilst the properties of this channel are consistent with it being a TASK-like potassium channel they do not precisely conform to those of either TASK-1 or TASK-3, 2) channel activity is highly dependent on cytosolic factors including ATP, and 3) changes in energy metabolism may play a role in regulating the activity of these background K+ channels.