Hydrogen currents through aconitine-modified sodium channels in the nerve fiber membrane

Ionic currents through aconitine-modified sodium channels of the Ranvier node membrane were measured by a voltage clamp method in an external medium free from sodium ions. A shift of pH of the solution below 4.6 led to the appearance of inward ionic currents, whose kinetics and activation region were characteristic of aconitine-modified sodium channels at low pH. These currents were blocked by the local anesthetic benzocaine in a concentration of 2 mM. Experiments with variation of the concentration of Ca⁺⁺, Tris⁺, TEA⁺, and choline⁺ in acid sodium-free solutions showed that these cations make no appreciable contribution to the inward current. It is concluded that the inward currents observed under these conditions are carried by H⁺ (or H₃O⁺) through aconitine-modified sodium channels. From the shifts of reversal potentials of the ionic currents the relative permeability (P_H/P_Na) for H⁺ was determined: 1059 ± 88. The results agree with the view that the aconitine-modified sodium channel is a relatively wide water pore, and that movement of H⁺ through it is limited by its binding with an acid group.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 508–516, September–October, 1982.     

Selectivity of edta-modified calcium channels to monovalent cations

The characteristics of slow inward sodium currents arising in response to membrane depolarization were studied in experiments on isolated dialyzed neurons of the snailHelix pomatia when the calcium-chelating agent EDTA was added to the calcium-free external solution. Values of the relative permeability of the corresponding ionic channels, determined from the shift of the equilibrium potential, were: P_Na+:P_Li+: +=1.00:0.80:0.55:0.21. The ratio between these values for "fast" sodium channels was 1.00:1.04:0.44:0.19. The induced sodium current was blocked by D-600 and nifedipine, which block calcium channels, more effectively than the calcium current of the same membrane (the corresponding dissociation constants were 10^–5 and 0.8·10^–5 mole/liter for the induced sodium current compared with 2.6·10^–5 and 2.3·10^–5 mole/liter for the calcium current). It is postulated on the basis of these data that the calcium channels have a principal selective filter similar to that of sodium channels, but also an additional binding site for bivalent cations, which prevents entry of monovalent cations into the channel. The addition of calcium-chelating agents to the calcium-free external solution liberates this site and thereby modifies the calcium channel into a sodium channel.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 491–498, September–October, 1982.     

Sodium-selective channels in membranes of rat macrophages

With the use of the patch-clamp technique, highly selective nonvoltage-gated sodium channels were found in the membrane of rat peritoneal macrophages. The inward single channel currents were measured in cell-attached and outside-out mode experiments at different holding membrane potentials within the range of-60 to +40 mV. The channels had a unitary conductance of 10.2 ± 0.2 pS with 145 mm Na⁺ in the external solution at 23–24°C. The results of ion-substitution experiments confirmed that this novel type of cation channel in macrophages is characterized by high selectivity for Na⁺ over K⁺ (as for Cs⁺, NH₄ ⁺, Ca²⁺, Ba²⁺) ions, whose conduction through these sodium-permeable channels was not measurable. Lithium is the only other ion that is transported by this pathway; the unitary conductance was equal to 3.9 ± 0.2 pS in the Li⁺-containing external solution. Single channel currents and conductance were found to be linearly dependent on the external sodium concentration. Sodium channels in macrophage membrane patches were not blocked by tetrodotoxin (0.01–1 m). Single sodium currents were reversibly inhibited by the external application of amiloride (0.1–2 mm) and its derivative ethylisopropilamiloride (0.01–0.1 Mm). The mechanism of channel block by amiloride and its analogue seems to be different.We thank Dr. G.N. Mozhayeva and Dr. A.P. Naumov for useful discussions. This work has been supported by a grant from the Russian Basic Research Foundation, 93-04-21722.     

Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

Selectivity and sensitivity to hydrogen ion blocking of batrachotoxin-modified sodium channels in nerve fiber membrane

Currents through batrachotoxin-modified sodium channels were measured by the voltage clamp method on the Ranvier node membrane. In experiments with replacement of Na⁺ in the external solution by K⁺ or NH ₄ ⁺ the following series of permeabilities, determined as reversal potentials according to the equation of a static field, was obtained — P_Na: \(P_{NH_4 }\) :P_K=1:0.47:0.19. The relative permeability for H⁺ was determined by measuring currents after replacement of Na⁺ in the external solution by nonpenetrating choline ions and lowering pH to 3.7–3.8. The ratio p_H/p_Na for sodium channels modified by batrachotoxin averaged 528±46. Modified channels were less sensitive to the blocking action of H⁺ than normal sodium channels. The difference in the effective values of pK of the acid group of normal and modified channels was 0.40–0.45.     

The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na⁺ ≈ Li⁺ > Tl⁺ > K⁺. The ratio P_K/P_Na is 1/12. The permeabilities to Rb⁺, Cs⁺, Ca⁺⁺, and Mg⁺⁺ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E_Na differs significantly from the Nernst potential for Na⁺ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria.     

Whole-cell and single-channel currents across the plasmalemma of corn shoot suspension cells

Summary Whole-cell sealed-on pipettes have been used to measure electrical properties of the plasmalemma surrounding protoplasts isolated from Black Mexican sweet corn shoot cells from suspension culture. In these protoplasts the membrane resting potential (V _m ) was found to be –59±23 mV (n=23) in 1mm K _o ^– . The meanV _m became more negative as [K^–] _o decreased, but was more positive than the K⁺ equilibrium potential. There was no evidence of electrogenic pump activity. We describe four features of the current-voltage characteristic of the plasmalemma of these protoplasts which show voltagegated channel activity. Depolarization of the whole-cell membrane from the resting potential activates time- and voltage-dependent outward current through K⁺-selective channels. A local minimum in the outward current-voltage curve nearV _m =150 mV suggests that these currents are mediated by two populations of K⁺-selective channels. The absence of this minimum in the presence of verapamil suggests that the activation of one channel population depends on the influx of Ca²⁺ into the cytoplasm. We identify unitary currents from two K⁺-selective channel populations (40 and 125 pS) which open when the membrane is depolarized; it is possible that these mediate the outward whole-cell current. Hyperpolarization of the membrane from the resting potential produces time- and voltage-dependent inward whole-cell current. Current activation is fast and follows an exponential time course. The current saturates and in some cases decreases at membrane potentials more negative than –175 mV. This current is conducted by poorly selective K⁺ channels, whereP _Cl/P _K=0.43±0.15. We describe a low conductance (20 pS) channel population of unknown selectivity which opens when the membrane is hyperpolarized. It is possible that these channels mediate inward whole-cell current. When the membrane is hyperpolarized to potentials more negative than –250 mV large, irregular inward current is activated. A third type of inward whole-cell current is briefly described. This activates slowly and with a U-shaped current-voltage curve over the range of membrane potentials –90<V _m <0 mV.     

Opposing Effects of Aluminum on Inward-Rectifier Potassium Currents in BeanRoot-Tip Protoplasts

Inward currents in root cap protoplasts of the aluminum-tolerant cultivar, Dade, of Phaseolus vulgaris L. were investigated using the whole-cell patch-clamp technique. The properties of these currents were similar to those seen in inward rectifying K⁺ channels in other plant tissues. Replacing bath K⁺ with Na⁺ nearly abolished the observed currents. Higher bath K⁺ concentrations increased inward currents. AlCl₃ in pH 4.7 bath solutions caused inward K⁺ currents to activate more rapidly and at more positive voltages when compared with AlCl₃ free solutions. In 10 μM AlCl₃ the activated inward K⁺ currents were significantly larger than in the AlCl₃-free solution at all voltages except at the most negative voltage of −174 mV and the least negative of −74 mV. In contrast, in 80 μM Al³⁺, when hyperpolarizing voltages were most negative, the inward K⁺ currents were inhibited relative to the currents in 10 μM AlCl₃. Enhancement of inward K⁺ currents by AlCl₃ is consistent with Al³⁺ binding to the external surface of the root cap protoplast, decreasing the surface charge, thus causing the channels to sense a more negative membrane potential. Inhibition of inward K⁺ currents with higher AlCl₃ concentrations and more negative voltages is consistent with Al³⁺ block of K⁺ channels.This revised version was published online in August 2005 with a corrected cover date.     

Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

Nitrate and chloride ions have different permeation pathways in skeletal muscle fibers ofRana pipiens

Summary The effects of pH on the permeability and conductance of the membranes to nitrate and to chloride of semitendinosus and lumbricalis muscle fibers were examined.Membrane potential responses to quick solution changes were recorded in semitendinosus fibers initially equilibrated in isotonic, high K₂SO₄ solutions. External solutions were first changed to ones in which either Rb⁺ or Cs⁺ replaced K⁺ and then to solutions containing either NO ₃ ^– or Cl^– to replace SO ₄ ^2– . The hyperpolarizations produced by Cl^– depend on external pH, being smaller in acid than in alkaline solutions. By contrast, hyperpolarizations produced by NO ₃ ^– were independent of external pH over a pH range from 5.5 to 9.0.In addition, voltage-clamp measurements were made on short lumbricalis muscle fibers. Initially they were equilibrated in isotonic solutions containing mainly K₂SO₄ plus Na₂SO₄. KCl or KNO₃ were added to the sulfate solutions and the fibers were equilibrated in these new solutions. When finally equilibrated the fibers had the same volume they had in the sulfate solutions before the additions. Constant hyperpolarizing voltage pulses of 0.6-sec duration were applied when all external K⁺ was replaced by TEA⁺. For these conditions, inward currents flowing during the voltage pulses were largely carried by Cl^– or NO ₃ ^– depending on the final equilibrating solution. Cl^– currents during voltage pulses were both external pH and time dependent. By contrast, NO ₃ ^– currents were independent of both external pH and time.The voltage dependence of NO ₃ ^– currents could be fit by constant field equations with aP _{NO 3} of 3.7·10^–6 cm/sec. The voltage dependence of the initial or instantaneous Cl^– currents at pH 7.5 and 9.0 could also be fit by constant field equations with P_Cl of 5.8·10^–6 and 7.9·10^–6 cm/sec, respectively. At pH 5.0, no measurable instantaneous Cl^– currents were found.From these results we conclude that NO ₃ ^– does not pass through the pH, time-dependent Cl^– channels but rather passes through a distinct set of channels. Furthermore, Cl^– ions do not appear to pass through the channels which allow NO ₃ ^– through. Consequently, the measured ratio ofP _Cl/P _{NO 3} based on membrane potential changes to ionic changes made on intact skeletal muscle fibers is not a measure of the selectivity of a single anion channel but rather is a measure of the relative amounts of different channel types.     

Chloride channels in human platelets: Evidence for activation by internal calcium

Summary Whole-cell patch-clamp recordings were made from freshly isolated human platelets. The pipette contained a high concentration of divalent cations, which permitted easy disruption of cell-attached membrane patches by suction. Single-channel currents were measured when the pipette contained isotonic BaCl₂ or MgCl₂ saline; over 30 sec –5 min an increasing number of channels appeared until conductance steps through individual channels could no longer be distinguished. The current-voltage relationship was curvilinear; chord conductance at –35 mV was 25 pS increasing to 45 to 52 pS at +45 mV. Ion substitution experiments showed the current to be primarily carried by Cl^–.E _rev was shifted 30 mV/10-fold change in external Cl^– (replaced by gluconate), was similar with BaCl₂ or MgCl₂ in the pipette and was not significantly shifted by replacing external Na⁺ with K⁺. Addition of 1mm BAPTA to the MgCl₂ pipette saline prevented activation of Cl^– currents; with isotonic CaCl₂ internal saline, current appeared immediately upon patch rupture, suggesting that the Cl^– channels are dependent on internal Ca²⁺, 5-nitro-2-(3-phenylpropylamino)-benzoate, reported to block a Cl^– conductance in studies of rat epithelial cells, caused a potent flickery block and may be a useful tool with which to investigate the physiological role of Cl^– currents in human platelets.     

Patch clamp studies on root cell vacuoles of a salt-tolerant and a salt-sensitive plantago species : regulation of channel activity by salt stress

Plantago media L. and Plantago maritima L. differ in their strategy toward salt stress, a major difference being the uptake and distribution of ions. Patch clamp techniques were applied to root cell vacuoles to study the tonoplast channel characteristics. In both species the major channel found was a 60 to 70 picosiemens channel with a low ion selectivity. The conductance of this channel for Na⁺ was the same as for K⁺, P_K⁺/P_Na⁺ = 1, whereas the cation/anion selectivity (P_K⁺/P_c1⁻) was about 5. Gating characteristics were voltage and calcium dependent. An additional smaller channel of 25 picosiemens was present in P. maritima. In the whole vacuole configuration, the summation of the single channel currents resulted in slowly activated inward currents (t^½ = 1.2 second). Inwardly directed, ATP-dependent currents could be measured against a ΔpH gradient of 1.5 units over the tonoplast. This observation strongly indicated the physiological intactness of the used vacuoles. The open probability of the tonoplast channels dramatically decreased when plants were grown on NaCl, although single channel conductance and selectivity were not altered.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

Modulation of Kir4.1 and Kir4.1-Kir5.1 channels by extracellular cations

This work demonstrates that extracellular Na⁺ modulates the cloned inwardly rectifying K⁺ channels Kir4.1 and Kir4.1-Kir5.1. Whole-cell patch clamp studies on astrocytes have previously indicated that inward potassium currents are regulated by external Na⁺. We expressed Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes to disclose if Kir4.1 and/or Kir4.1-Kir5.1 at the molecular level are responsible for the observed effect of [Na⁺]_o and to investigate the regulatory mechanism of external cations further. Our results showed that Na⁺ has a biphasic modulatory effect on both Kir4.1 and Kir4.1-Kir5.1 currents. Depending on the Na⁺-concentration and applied voltage, the inward Kir4.1/Kir4.1-Kir5.1 currents are either enhanced or reduced by extracellular Na⁺. The Na⁺ activation was voltage-independent, whereas the Na⁺-induced reduction of the Kir4.1 and Kir4.1-Kir5.1 currents was both concentration-, time- and voltage-dependent. Our data indicate that the biphasic effect of extracellular Na⁺on the Kir4.1 and Kir4.1-Kir5.1 channels is caused by two separate mechanisms.     

Ion channels and ATP-driven pumps involved in ion transport across the tonoplast of sugarbeet vacuoles

The electrical properties of the tonoplast of mature sugarbeet root vacuoles have been studied using the patch-clamp technique. In whole-vacuole recordings, the addition of 5 mM Mg-ATP to the external solution activated a proton-translocating ATPase which produced inward currents of up to 65 pA. Furthermore, we identified a voltage-dependent membrane conductance which activated at hyperpolarized (inside-negative) potentials and decreased at positive potentials. Outside-out membrane patches predominantly contained a channel which showed an increasing probability of opening at potentials more negative than about –20 mV. These channels can account for the macroscopic currents recorded in whole vacuoles. The permeability sequence of the channel for cations and anions was: P_K^staggered+ = P_Na⁺ >P_Ac⁻ >P_NO3⁻ >P_Mal²⁻ >P_Cl⁻. The unit conductance of this channel was about 70 pS in symmetrical 50 mM KCl and 180 pS in symmetrical 200 mM KCl solutions. Another channel type of smaller conductance (15 pS in 50 mM KCl) was also present, but its properties have not yet been studied. The permeability sequence of the nonselective channel corresponds to that found by tracer measurements in vacuole suspensions, implying that the channel studied may present the molecular pathway for the movement of ions across the tonoplast.     

Satellite glial cells In Situ within mammalian prevertebral ganglia express K+ channels active at rest potential

Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (–79.1±1.2 mV) was found to be very nearly the same as the K⁺ reversal potential and 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K⁺, Na⁺ and gluconate. Only K⁺-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K⁺]₀) were detected. These channels were active (P ₀=0.7–0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K⁺] in the pipette. Cl^–-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches.The properties of the K⁺ channels (increased inward current with [K⁺] and detectable outward current at low [K⁺]) are well suited for siphoning the K⁺ released by active neurons.     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.     

Predominance of poorly reopening single Na+ channels and lack of slow Na+ inactivation in neonatal cardiocytes

Summary Elementary Na⁺ currents through single cardiac Na⁺ channels were recorded at –50 mV in cell-attached patches from neonatal rat cardiocytes kept at holding potentials between –100 and –120 mV.Na⁺ channel activity may occur as burst-like, closely-timed repetitive openings with shut times close to 0.5–0.6 msec, indicating that an individual Na⁺ channel may reopen several times during step depolarization. A systematic quantiative analysis in 19 cell-attached patches showed that reopening may be quite differently pronounced. The majority, namely 16 patches, contained Na⁺ channels with a low tendency to reopen. This was evidenced from the average value for the mean number of openings per sequence, 2.5. Strikingly different results were obtained in a second group of three patches. Here, a mean number of openings per sequence of 3.42, 3.72, and 5.68 was found. Ensemble averages from the latter group of patches revealed macroscopic Na⁺ currents with a biexponential decay phase. Reconstructed Na⁺ currents from patches with poorly reopening Na⁺ channels were devoid of a slow decay component. This strongly suggests that reopening may be causally related to slow Na⁺ inactivation. Poorly pronounced reopening and, consequently, the lack of slow Na⁺ inactivation could be characteristic features of neonatal cardiac Na⁺ channels.     

Novel actions of ryanodine and analogues—Perturbers of potassium channels

The effects of ryanodine, 9,21-didehydroryanodine and 9,21-didehydroryanodol on two types of K⁺ channel (a maxi, Ca²⁺-activated, 170 pS channel (BK channel) and an inward rectifier, stretch-sensitive channel of 35 pS conductance (IK channel) found in the plasma membrane of locust skeletal muscle have been investigated. 10^–9M-10^–5M ryanodine irreversibly induced a dose-dependent reduction of the reversal potential (V_rev) of the currents of both channels, i.e. from 60 mV in the absence of the alkaloid to 15 mV for 10^–5M ryanodine, measured under physiologically normal K⁺ and Na⁺ gradients. In both cases the change in the ionic selectivity was Ca²⁺-independent. 9,21-didehydroryanodine and 9,21-didehyroryanodol also reduced V_rev, but only to 35 mV during application of 10^–5M of these compounds. Additionally, 9,21-didehydroryanodine reversibly diminished the conductances of the two K⁺ channels. To test the hypothesis that ryanoids increase Na⁺ permeability by enlarging the K⁺ channels, the channels were probed with quaternary ammonium ions during ryanoid application. When applied to the cytoplasmic face of inside-out patches exised from locust muscle membrane, TEA blocked the K⁺ channels in a voltage-dependent fashion. The dissociation constant (K_d(0)) for TEA block of the IK channel was reduced from 44 mM to 1 mM by 10^–7 M ryanodine, but the voltage-dependence of the block was unaffected. Qualitatively similar data were obtained for the BK channel. Ryanodine had no effect on the K_d for cytoplasmically-applied TMA. However, the voltage-dependence for TMA block was increased for both K⁺ channels, from 0.47 to 0.8 with 10^–6M ryanodine. The effects of ryanodine on TEA and TMA block support the hypothesis that ryanodine enlarges the K⁺ channels so as to facilitate permeation of partially hydrated Na⁺ ions.