On the Gating Mechanisms of the Light-dependent Conductance in Pecten Hyperpolarizing Photoreceptors: Does Light Remove Inactivation in Voltage-dependent K Channels?

The hyperpolarizing receptor potential of ciliary photoreceptors of scallop and other mollusks is mediated by a cGMP-activated K conductance; these cells also express a transient potassium current triggered by depolarization. During steady illumination, the outward currents elicited by voltage steps lose their decay kinetics. One interesting conjecture that has been proposed is that the currents triggered by light and by depolarization are mediated by the same population of channels, and that illumination evokes the receptor potential by removing their steady-state inactivation. Exploiting the information that has become available on the phototransduction cascade of ciliary photoreceptors, we demonstrated that the same downstream signaling elements are implicated in the modulation of voltage-elicited currents: direct chemical stimulation both at the level of the G protein and of the final messenger that controls the light-dependent channels (cGMP) also attenuate the falling phase of the voltage-activated current. Application of a protein kinase G antagonist was ineffective, suggesting that a cGMP-initiated phosphorylation step is not implicated. To ascertain the commonality of ionic pathways we used pharmacological blockers. Although millimolar 4-aminopyridine (4-AP) suppressed both currents, at micromolar concentrations only the photocurrent was blocked. Conversely, barium completely and reversibly antagonized the transient voltage-activated current with no detectable effect on the light-evoked current. These results rule out that the same ionic pores mediate both currents; the mechanism of light modulation of the depolarization-evoked K current was elucidated as a time-dependent increase in the light-sensitive conductance that is superimposed on the inactivating K current.     

Detection of potassium currents and regulation of multidrug resistance by potassium channels in human gastric cancer cells

The present study aimed to investigate the potassium currents and further explore the role of potassium channels in drug response of gastric cancer cells. By patch-clamp technique, potassium currents of human gastric cancer cell SGC7901 were recorded in the mode of voltage clamp. Both 4-aminopyridine (4-AP) and tetraethylammonium (TEA) could almost completely block this current. The chemotherapeutic drugs, adriamycin or 5-fluorouracil could significantly increase the K(+) current density on SGC7901 cells in a dose-dependent manner. 4-AP or TEA was found to restrain adriamycin-induced apoptosis and enhance multidrug-resistant phenotype of SGC7901 cells. Up-regulation of Kv1.5, which has been found widely expressed in gastric cancer cells including SGC7901, increased the K(+) current density and sensitivity of SGC7901 cells to multiple chemotherapeutic drugs, whereas down-regulation of Kv1.5 enhanced the drug-resistant phenotype of SGC7901 cells. In conclusion, potassium channels may exert regulatory effects on multidrug resistance by regulating drug-induced apoptosis in gastric cancer cells.     

Two types of potassium current in rabbit cultured Schwann cells

Voltage-gated outward currents were studied in rabbit cultured Schwann cells with the 'whole-cell' configuration of the patch-clamp method. Four components of such currents were identified. The first, which was abolished by replacement of the external chloride ions by the large impermeant anion gluconate, was identified as a chloride current. The second and third were identified as potassium currents. One type of potassium current was reduced substantially by either 4-aminopyridine (4-AP) or tetraethylammonium ion (TEA). Its sensitivity to blocking by 4-AP was highly voltage-dependent: the equilibrium dissociation constant (K) was threefold greater when measured at +10 mV than when measured at -40 mV (where it was about 80 microM). The second type of potassium current was relatively insensitive to 4-AP, but was blocked by TEA. The TEA sensitivity of the two types of potassium currents was similar and displayed no obvious voltage-dependence (K approximately 200 microM). The fourth component of current was not reduced by 4-AP or TEA at concentrations less than 10 mM. Whether or not this last component is a potassium current is unclear.     

Properties of tetraethylammonium ion-resistant K+ channels in the photoreceptor membrane of the giant barnacle

After the offset of illumination, barnacle photoreceptors undergo a large hyperpolarization that lasts seconds or minutes. We studied the mechanisms that generate this afterpotential by recording afterpotentials intracellularly from the medial photoreceptors of the giant barnacle Balanus nubilus. The afterpotential has two components with different time-courses: (a) an earlier component due to an increase in conductance to K+ that is not blocked by extracellular tetraethylammonium ion (TEA+) or 3-aminopyridine (3-AP) and (b) a later component that is sensitive to cardiac glycosides and that requires extracellular K+, suggesting that it is due to an electrogenic Na+ pump. The K+ conductance component increases in amplitude with increasing CA++ concentration and is inhibited by extracellular Co++; the Co++ inhibition can be overcome by increasing the Ca++ concentration. Thus, the K+ conductance component is Ca++ dependent. An afterpotential similar to that evoked by a brief flash of light is generated by depolarization with current in the dark and by eliciting Ca++ action potentials in the presence of TEA+ in the soma, axon, or terminal regions of the photoreceptor. The action potential undershoot is generated by an increase in conductance to K+ that is resistant to TEA+ and 3-AP and inhibited by Co++. The similarity in time-course and pharmacology of the hyperpolarization afterpotentials elicited by (a) a brief flash of light, (b) depolarization with current, and (c) an action potential indicates that Ca++-dependent K+ channels throughout the photoreceptor membrane are responsible for all three hyperpolarizing events.     

The role of slow potassium current in phenomena of voltage-dependent action of 4-aminopyridine in amphibian myelinated nerve fibres

The mechanism underlying the voltage-dependent action of 4-aminopyridine (4-AP) is investigated in experiments on amphibian myelinated nerve fibres (Rana ridibunda Pallas) by way of extracellular recording of electrical activity and using activators of potassium current (potassium-free solution and nitric oxide NO) and inhibitors of sodium current (tetrodotoxin). Measurement of action potential (AP) areas was used to evaluate the extent of general membrane depolarization during the activity of nerve fibres. Tetrodotoxin-induced decrease in general membrane depolarization (when the action potential amplitude was reduced by less than 20%) leads to an increase in the duration of depolarizing after-potential (DAP). This supports the dependence of time course of DAP in the presence of 4-AP on ratio of fast and slow potassium channels. In the absence of 4-AP, potassium-free solution and NO increase the potassium current through fast potassium channels (decreasing AP duration, reducing DAP and sometimes producing fast hyperpolarizing after-potential (HAP) after shortened AP), and in the presence of 4-AP these activators increase potassium current through unblocked slow potassium channels (making the development of slow HAP induced by 4-AP more rapid). The increase of slow HAP induced by 4-AP under the influence of potassium-free solution with NO supports the idea that slow HAP is due to activation of slow potassium channels and argues against the notion of removal of block of fast potassium channels. All analyzed phenomena of voltage-dependent action of 4-AP in amphibian myelinated nerve fibers can be accounted for by the activation of slow potassium current produced by membrane depolarization and a decrease of the amount of fast potassium channels involved in the membrane repolarization.     

Two light-dependent conductances in Lima rhabdomeric photoreceptors

Light-dependent membrane currents were recorded from solitary Lima photoreceptors with the whole-cell clamp technique. Light stimulation from a holding voltage near the cell's resting potential evokes a transient inward current graded with light intensity, accompanied by an increase in membrane conductance. While the photocurrent elicited by dim flashes decays smoothly, at higher stimulus intensities two kinetically distinct components become visible. Superfusion with TEA or intracellular perfusion with Cs do not eliminate this phenomenon, indicating that it is not due to the activation of the Ca-sensitive K channels that are present in these cells. The relative amplitude of the late component vs. the early peak of the light response is significantly more pronounced at -60 mV than at -40 mV. At low light intensities the reversal potential of the photocurrent is around 0 mV, but with brighter lights no single reversal potential is found; rather, a biphasic response with an inward and an outward component can be seen within a certain range of membrane voltages. Light adaptation through repetitive stimulation with bright flashes diminishes the amplitude of the early but not the late phase of the photocurrent. These observations can be accounted for by postulating two separate light-dependent conductances with different ionic selectivity, kinetics, and light sensitivity. The light response is also shown to interact with some of the voltage-sensitive conductances: activation of the Ca current by a brief conditioning prepulse is capable of attenuating the photocurrent evoked by a subsequent test flash. Thus, Ca channels in these cells may not only shape the photoresponse, but also participate in the process of light adaptation.     

Differential blockage of two types of potassium channels in the crab giant axon

Summary Measurements were made of the kinetic and steady-state characteristics of the potassium conductance in the giant axon of the crabsCarcinus maenas andCancer pagirus. The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate types of potassium channels exist in these axons (M. E. Quinta-Ferreira, E. Rojas and N. Arispe,J. Membrane Biol. 66:171–181, 1982). It is shown here that, with small concentrations of conventional K⁺-channel blockers, it is possible to differentially inhibit these channels. The potassium channels with activation and fast inactivation gating (m³h, Hodgkin-Huxley kinetics) were blocked by external application of 4 amino-pyridine (4-AP). The potassium channels with standard gating (n⁴, Hodgkin-Huxley kinetics) were preferentially inhibited by externally applied tetraethylammonium (TEA). The differential blockage of the two types of potassium conductance changes suggests that they represent two different populations of potassium channels.It is further shown here that blocking the early transient conductance increase leads to the inhibition of the repetitive electrical activity induced by constant depolarizing current injection in fibers fromCardisoma guanhumi.     

Voltage-dependent calcium and calcium-activated potassium currents of a molluscan photoreceptor

Two-microelectrode voltage clamp studies were performed on the somata of Hermissenda Type B photoreceptors that had been isolated by axotomy from all synaptic interaction as well as any impulse-generating (i.e., active) membrane. In the presence of 2-10 mM 4-aminopyridine (4-AP) and 100 mM tetraethylammonium ion (TEA), which eliminated two previously described voltage-dependent potassium currents (IA and the delayed rectifier), a voltage-dependent outward current was apparent in the steady state responses to command voltage steps more positive than -40 mV (absolute). This current increased with increasing external Ca++. The magnitude of the outward current decreased and an inward current became apparent following EGTA injection. Substitution of external Ba++ for Ca++ also made the inward current more apparent. This inward current, which was almost eliminated after being exposed for approximately 5 min to a solution in which external Ca++ was replaced with Cd++, was maximally activated at approximately 0 mV. Elevation of external potassium allowed the calcium (ICa++) and calcium-dependent K+ (IC) currents to be substantially separated. Command pulses to 0 mV elicited maximal ICa++ but no IC because no K+ currents flowed at their new reversal potential (0 mV) in 300 mM K+. At a holding potential of -60 mV, which was now more negative than the potassium equilibrium potential, EK+, in 300 mM K+, IC appeared as an inward tail current after positive command steps. The voltage dependence of ICa++ was demonstrated with positive steps in 100 mM Ba++, 4-AP, and TEA. Other data indicated that in 10 mM Ca++, IC underwent pronounced and prolonged inactivation whereas ICa++ did not. When the photoreceptor was stimulated with a light step (with the membrane potential held at -60 mV), there was also a prolonged inactivation of IC. In elevated external Ca++, ICa++ also showed similar inactivation. These data suggest that IC may undergo prolonged inactivation due to a direct effect of elevated intracellular Ca++, as was previously shown for a voltage-dependent potassium current, IA. These results are discussed in relation to the production of training-induced changes of membrane currents on retention days of associative learning.     

The light-sensitive conductance of hyperpolarizing invertebrate photoreceptors: a patch-clamp study

Tight-seal recording was employed to investigate membrane currents in hyperpolarizing ciliary photoreceptors enzymatically isolated from the eyes of the file clam (Lima scabra) and the bay scallop (Pecten irradians). These two organisms are unusual in that their double retinas also possess a layer of depolarizing rhabdomeric cells. Ciliary photoreceptors from Lima have a rounded soma, 15-20 microns diam, and display a prominent bundle of fine processes up to 30 microns long. The cell body of scallop cells is similar in size, but the ciliary appendages are modified, forming small spherical structures that protrude from the cell. In both species light stimulation at a voltage near the resting potential gives rise to a graded outward current several hundred pA in amplitude, accompanied by an increase in membrane conductance. The reversal potential of the photocurrent is approximately -80 mV, and shifts in the positive direction by approximately 39 mV when the concentration of extracellular K is increased from 10 to 50 mM, consistent with the notion that light activates K-selective channels. The light-activated conductance increases with depolarization in the physiological range of membrane voltages (-30 to -70 mV). Such outward rectification is greatly reduced after removal of divalent cations from the superfusate. In Pecten, cell- attached recordings were also obtained; in some patches outwardly directed single-channel currents could be activated by light but not by voltage. The unitary conductance of these channels was approximately 26 pS. Solitary ciliary cells also gave evidence of the post stimulus rebound, which is presumably responsible for initiating the "off" discharge of action potentials at the termination of a light stimulus: in patches containing only voltage-dependent channels, light stimulation suppressed depolarization-induced activity, and was followed by a strong burst of openings, directly related to the intensity of the preceding photostimulation.     

Divalent cation interactions with light-dependent K channels. Kinetics of voltage-dependent block and requirement for an open pore.

The light-dependent K conductance of hyperpolarizing Pecten photoreceptors exhibits a pronounced outward rectification that is eliminated by removal of extracellular divalent cations. The voltage-dependent block by Ca(2+) and Mg(2+) that underlies such nonlinearity was investigated. Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca(2+) than Mg(2+) (K(1/2) approximately 16 and 61 mM, respectively, at V(m) = -30 mV). Neither cation is measurably permeant. Manipulating the concentration of permeant K ions affects the blockade, suggesting that the mechanism entails occlusion of the permeation pathway. The voltage dependency of Ca(2+) block is consistent with a single binding site located at an electrical distance of delta approximately 0.6 from the outside. Resolution of light-dependent single-channel currents under physiological conditions indicates that blockade must be slow, which prompted the use of perturbation/relaxation methods to analyze its kinetics. Voltage steps during illumination produce a distinct relaxation in the photocurrent (tau = 5-20 ms) that disappears on removal of Ca(2+) and Mg(2+) and thus reflects enhancement or relief of blockade, depending on the polarity of the stimulus. The equilibration kinetics are significantly faster with Ca(2+) than with Mg(2+), suggesting that the process is dominated by the "on" rate, perhaps because of a step requiring dehydration of the blocking ion to access the binding site. Complementary strategies were adopted to investigate the interaction between blockade and channel gating: the photocurrent decay accelerates with hyperpolarization, but the effect requires extracellular divalents. Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent. These observations suggest that equilibration of block at different voltages requires an open pore. Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.     

Anomalous effects of external TEA on permeation and gating of the A-type potassium current in H. aspersa neuronal somata

The model proposed for external TEA block of Shaker K+ channels predicts a proportional relationship between TEA sensitivity and calculated electrical distance derived from measurements of voltage dependence of TEA block. In the present study, we examined this relationship for the A-type K+ current (IA) of Helix aspersa in neuronal somata using the whole-cell patch-clamp technique. External TEA inhibited IA with strong voltage dependence, such that the TEA dissociation constant was increased at depolarized test potentials. The half-inhibition constant (V0.5) for TEA block was approximately 21 mM at 0 mV, and V0.5 increased to approximately 67 mM at 50 mV. The calculated electrical distance for TEA block suggested that TEA traversed 65% of the way into the membrane electrical field. TEA also caused significant shifts in the voltage-dependence of A-type K+ channel gating. For example, at TEA concentrations below that required to fully suppress delayed outward currents, TEA caused depolarizing shifts in the voltage-dependence of A-type channel activation, steady-state inactivation, time for removal of inactivation, and slowed channel activation kinetics. Taken together, these observations suggest that TEA biased the local field potential near voltage-sensing domains of A-type K+ channels, causing the transmembrane electrical field to be relatively hyperpolarized in the presence of TEA. In summary, the calculated electrical distance of TEA block of A-type K+ channels in H. aspersa neurons is unprecedented among other K+ channels. This raises concerns about the conventional interpretation of this value. Furthermore, the voltage-dependent properties of IA are modified by TEA at concentrations previously used to isolate delayed rectifier potassium channels (IKDR) selectively. This lack of specificity has important implications for recent, as well as future studies of IA in H. aspersa and possibly other snail neurons.     

Aminopyridine block of transient potassium current

The blocking action of 4-aminopyridine (4-AP) and 3, 4-diaminopyridine (Di-AP) on transient potassium current (IA) in molluscan central neurons was studied in internal perfusion voltage-clamp experiments. Identical blocking effects were seen when the drugs were applied either externally or internally. It was found that aminopyridines have two kinds of effects on IA channels. The first involves block of open channels during depolarizing pulses and results in a shortening of the time to peak current and an increase in the initial rate of decay of current. This effect of the drug is similar to the block of delayed potassium current by tetraethylammonium (TEA). The other effect is a steady block that increases in strength during hyperpolarization, is removed by depolarization, and is dependent on the frequency of stimulation. The voltage dependence of steady state block approximates the voltage dependence of inactivation gating a changes e-fold in approximately 10 mV. These data suggest that the strength of block may depend on the state of IA gating such that the resting state of the channel with open inactivation gate is more susceptible to block than are the open or inactivated states. A multistate sequential model for IA gating and voltage-dependent AP block is developed.     

Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia sensory neurons

The effects of a variety of K+ channel blockers on current flow through single serotonin-sensitive K+ channels (the S channels) of Aplysia sensory neurons were studied using the patch-clamp technique. Tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Co2+ and Ba2+ were first applied to the external membrane surface using cell-free outside-out patches. At concentrations up to 10 mM, these agents had little or no effect on single S-channel currents. At higher concentrations, external TEA acted as a fast open-channel blocker, reducing the single-channel current amplitude according to a simple one-to-one binding scheme with an apparent Kd of 90 mM. Blockage by external TEA is voltage independent. Internal TEA also acts as an open-channel blocker, with an apparent Kd of approximately 40 mM and a relatively weak voltage dependence, corresponding to an apparent electrical distance to the internal TEA-binding site of 0.1. Both internal and external TEA block the open channel selectively, with an affinity that is 10-100-fold greater than the affinity for the closed channel. Internal Ba2+ acts as a slow channel blocker, producing long closures of the channel, and binding with an apparent Kd of approximately 25-30 microM. These results show that single S-channel currents share a similar pharmacological profile with the macroscopic S current previously characterized with voltage clamp. On the basis of these results, a structural model for S-channel opening is proposed.     

Characterization of slowly inactivating KV{alpha} current in rabbit pulmonary neuroepithelial bodies: effects of hypoxia and nicotine

Pulmonary neuroepithelial bodies (NEB) form innervated cell clusters that express voltage-activated currents and function as airway O(2) sensors. We investigated A-type K(+) currents in NEB cells using neonatal rabbit lung slice preparation. The whole cell K(+) current was slowly inactivating with activation threshold of approximately -30 mV. This current was blocked approximately 27% by blood-depressing substance I (BDS-I; 3 microM), a selective blocker of Kv3.4 subunit, and reduced approximately 20% by tetraethylammonium (TEA; 100 microM). The BDS-I-sensitive component had an average peak value of 189 +/- 14 pA and showed fast inactivation kinetics that could be fitted by one-component exponential function with a time constant of (tau1) 77 +/- 10 ms. This Kv slowly inactivating current was also blocked by heteropodatoxin-2 (HpTx-2; 0.2 microM), a blocker of Kv4 subunit. The HpTx-2-sensitive current had an average peak value of 234 +/- 23 pA with a time constant (tau) 82 +/- 11 ms. Hypoxia (Po(2) = 15-20 mmHg) inhibited the slowly inactivating K(+) current by approximately 47%, during voltage steps from -30 to +30 mV, and no further inhibition occurred when TEA was combined with hypoxia. Nicotine at concentrations of 50 and 100 microM suppressed the slowly inactivating K(+) current by approximately 24 and approximately 40%, respectively. This suppression was not reversed by mecamylamine suggesting a direct effect of nicotine on these K(+) channels. In situ hybridization experiments detected expression of mRNAs for Kv3.4 and Kv4.3 subunits, while double-label immunofluorescence confirmed membrane localization of respective channel proteins in NEB cells. These studies suggest that the hypoxia-sensitive current in NEB cells is carried by slowly inactivating A-type K(+) channels, which underlie their oxygen-sensitive potassium currents, and that exposure to nicotine may directly affect their function, contributing to smoking-related lung disease.     

The light-sensitive conductance of melanopsin-expressing Joseph and Hesse cells in amphioxus

Two types of microvillar photoreceptors in the neural tube of amphioxus, an early chordate, sense light via melanopsin, the same photopigment as in "circadian" light detectors of higher vertebrates. Because in amphioxus melanopsin activates a G(q)/phospholipase C cascade, like phototransduction in arthropods and mollusks, possible commonalities in the photoconductance were investigated. Unlike other microvillar photoreceptors, reversal of the photocurrent can only be attained upon replacement of extracellular Na(+). In addition to Na(+), Ca(2+) is also permeant, as indicated by the fact that (a) in normal ionic conditions the photocurrent remains inward at V(m) > E(Na); (b) in Na-free solution a small residual inward photocurrent persists at V(m) near resting level, provided that Ca is present; and (c) V(rev) exhibits a modest shift with [Ca](o) manipulations. The unusual reversal is accounted for by an uncommonly low permeability of the light-dependent channels to K(+), as [K](o) only marginally affects the photocurrent amplitude and its reversal. Lanthanum and ruthenium red (RuR), two TRP channel antagonists, reversibly suppress the response to photostimulation of moderate intensity; therefore, the melanopsin-initiated cascade may recruit ion channels of the same family as those of rhabdomeric photoreceptors. With brighter lights, blockage declines, so that both La(3+) and RuR induce a right shift in the sensitivity curve without a reduction of its asymptote. Nonetheless, an effect on the transduction cascade, rather than the channels, was ruled out on the basis of the voltage dependency of the blockade and the lack of effects of intracellular application of the same substances. The mechanisms of action of these antagonists thus entail a state-dependent blockade, with a higher affinity for the channel in the closed conformation. Collectively, the results indicate a kinship of the light-sensitive channels of amphioxus with those of invertebrate rhabdomeric visual cells and support the representation of this lineage of photoreceptors among chordates.     

Electrophysiological Evidence for Intrinsic Pacemaker Currents in Crayfish Parasol Cells

I used sharp intracellular electrodes to record from parasol cells in the semi-isolated crayfish brain to investigate pacemaker currents. Evidence for the presence of the hyperpolarization-activated inward rectifier potassium current was obtained in about half of the parasol cells examined, where strong, prolonged hyperpolarizing currents generated a slowly-rising voltage sag, and a post-hyperpolarization rebound. The amplitudes of both the sag voltage and the depolarizing rebound were dependent upon the strength of the hyperpolarizing current. The voltage sag showed a definite threshold and was non-inactivating. The voltage sag and rebound depolarization evoked by hyperpolarization were blocked by the presence of 5–10 mM Cs²⁺ ions, 10 mM tetraethyl ammonium chloride, and 10 mM cobalt chloride in the bathing medium, but not by the drug ZD 7288. Cs⁺ ions in normal saline in some cells caused a slight increase in mean resting potential and a reduction in spontaneous burst frequency. Many of the neurons expressing the hyperpolarization-activated inward potassium current also provided evidence for the presence of the transient potassium current I_A, which was inferred from experimental observations of an increased latency of post-hyperpolarization response to a depolarizing step, compared to the response latency to the depolarization alone. The latency increase was reduced in the presence of 4-aminopyridine (4-AP), a specific blocker of I_A. The presence of 4-AP in normal saline also induced spontaneous bursting in parasol cells. It is conjectured that, under normal physiological conditions, these two potassium currents help to regulate burst generation in parasol cells, respectively, by helping to maintain the resting membrane potential near a threshold level for burst generation, and by regulating the rate of rise of membrane depolarizing events leading to burst generation. The presence of post-burst hyperpolarization may depend upon I_A channels in parasol cells.     

Voltage-dependence of ion permeation in cyclic GMP-gated ion channels is optimized for cell function in rod and cone photoreceptors

The kinetics of the photocurrent in both rod and cone retinal photoreceptors are independent of membrane voltage over the physiological range (-30 to -65 mV). This is surprising since the photocurrent time course is regulated by the influx of Ca(2+) through cGMP-gated ion channels (CNG) and the force driving this flux changes with membrane voltage. To understand this paradigm, we measured Pf, the fraction of the cyclic nucleotide-gated current specifically carried by Ca(2+) in intact, isolated photoreceptors. To measure Pf we activated CNG channels by suddenly increasing free 8-Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca(2+) binding dye, Fura-2, also loaded into the cells. We determined Pf under physiological solutions at various holding membrane voltages between -65 and -25 mV. Pf is larger in cones than in rods, but in both photoreceptor types its value is independent of membrane voltage over the range tested. This biophysical feature of the CNG channels offers a functional advantage since it insures that the kinetics of the phototransduction current are controlled by light, and not by membrane voltage. To explain our observation, we developed a rate theory model of ion permeation through CNG channels that assumes the existence of two ion binding sites within the permeation pore. To assign values to the kinetic rates in the model, we measured experimental I-V curves in membrane patches of rods and cones over the voltage range -90 to 90 mV in the presence of simple biionic solutions at different concentrations. We optimized the fit between simulated and experimental data. Model simulations describe well experimental photocurrents measured under physiological solutions in intact cones and are consistent with the voltage-independence of Pf, a feature that is optimized for the function of the channel in photoreceptors.     

Interaction of internal anions with potassium channels of the squid giant axon

The interaction of internal anions with the delayed rectifier potassium channel was studied in perfused squid axons. Changing the internal potassium salt from K+ glutamate- to KF produced a reversible decline of outward K currents and a marked slowing of the activation of K channels at all voltages. Fluoride ions exert a differential effect upon K channel gating kinetics whereby activation of IK during depolarizing steps is slowed dramatically, but the rate of closing after the step is not much altered. These effects develop with a slow time course (30-60 min) and are specific for K channels over Na channels. Both the amplitude and activation rate of IK were restored within seconds upon return to internal glutamate solutions. The fluoride effect is independent of the external K+ concentration and test membrane potential, and does not recover with repetitive application of depolarizing voltage steps. Of 11 different anions tested, all inorganic species induced similar decreases and slowing of IK, while K currents were maintained during extended perfusion with several organic anions. Anions do not alter the reversal potential or shape of the instantaneous current-voltage relation of open K channels. The effect of prolonged exposure to internal fluoride could be partially reversed by the addition of cationic K channel blocking agents such as TEA+, 4-AP+, and Cs+. The competitive antagonism between inorganic anions and internal cationic K channel blockers suggests that they may interact at a related site(s). These results indicate that inorganic anions modify part of the K channel gating mechanism (activation) at a locus near the inner channel surface.     

Dynamics of aminopyridine block of potassium channels in squid axon membrane

Aminopyridines (2-AP, 3-AP, and 4-AP) selectively block K channels of squid axon membranes in a manner dependent upon the membrane potential and the duration and frequency of voltage clamp pulses. They are effective when applied to either the internal or the external membrane surface. The steady-state block of K channels by aminopyridines is more complete for low depolarizations, and is gradually relieved at higher depolarizations. The K current in the presence of aminopyridines rises more slowly than in control, the change being more conspicuous in 3-AP and 4-AP than in 2-AP. Repetitive pulsing relieves the block in a manner dependent upon the duration and interval of pulses. The recovery from block during a given test pulse is enhanced by increasing the duration of a conditioning depolarizing prepulse. The time constant for this recovery is in the range of 10-20 ms in 3-AP and 4-AP, and shorter in 2-AP. Twin pulse experiments with variable pulse intervals have revealed that the time course for re-establishment of block is much slower in 3-AP and 4-AP than in 2-AP. These results suggest that 2-AP interacts with the K channel more rapidly than 3-AP and 4-AP. The more rapid interaction of 2-AP with K channels is reflected in the kinetics of K current which is faster than that observed in 3-AP or 4-AP, and in the pattern of frequency-dependent block which is different from that in 3-AP or 4-AP. The experimental observations are not satisfactorily described by alterations of Hodgkin-Huxley n-type gating units. Rather, the data are consistent with a simple binding scheme incorporating no changes in gating kinetics which conceives of aminopyridine molecules binding to closed K channels and being released from open channels in a voltage-dependent manner.