The pharmacological characteristics of two types of cholinoreceptors in the membrane of dialyzed neurons

1. The ramped voltage clamp technique was developed as a rapid means of studying the effects of certain nicotinic and muscarinic agents on ionic involvement and conductance changes during acetylcholine (ACh) responses of Helix pomatia neurons. 2. Atropine was found to be a potent cholinolytic on A-type neurons, ACh responses of which are blocked by ouabain and mediated by Na+ and Cl- permeabilities, while d-tubocurarine blocked B-type ACh responses which are insensitive to ouabain and mediated by Na+ and K+ permeabilities. 3. Nicotinic agent butyrylcholine was found to be a potent cholinomimetric on B-type cells. 4. The results suggest that ACh receptors on A-type cells are more "muscarinic" while those on B-type cells are more "nicotinic". 5. It was also suggested that both muscarinic and nicotinic ACh receptors may coexist in the Helix neuronal membrane and the possibility of ACh interacting with one of them is determined by the level of phosphorylation of the membrane proteins.     

Studies of insulin action on the amphibian oocyte plasma membrane using NMR, electrophysiological and ion flux techniques

Insulin (0.1-10 microM) reinitiates the meiotic divisions in Rana oocytes and produces a 14-20 mV negative-going hyperpolarization of the plasma membrane as well as a 0.25 unit increase in intracellular pH during the first 90 min. During hyperpolarization, the Na+ conductance of the membrane decreases by 40-50% with a concomitant increase in 22Na+ uptake from the medium. The increased uptake of Na+ during a period of decreasing Na+ conductance is apparently due to an increase in fluid phase turnover associated with insulin-mediated endocytosis. Both membrane hyperpolarization and increase in pHi are Na+-dependent and are blocked by the serine proteinase inhibitor, phenylmethylsulfonyl fluoride. The membrane potential of the prophase oocyte has a significant electrogenic component with potential but not conductance sensitive to glycosides and substitution of Li+ for Na+. Insulin hyperpolarizes Li+ or glycoside-treated oocytes whereas glycosides do not affect insulin-hyperpolarized oocytes. [3H]Ouabain binding by the plasma membrane of the untreated oocyte shows at least two K+-sensitive components (Kd = 42 and 2000 nM) linked to inhibition of the Na+ pump. Insulin-treated oocytes show a single class of intermediate-affinity ouabain sites (Kd = 490 nM) which appear to result from insulin-induced internalization of membrane-bound ouabain. [125I]Insulin binding to the plasma membrane shows a class of high-affinity sites (Kd = 87 nM) with 40-50 pump sites per insulin-binding site. Our results suggest that insulin-induced mediator peptides stimulate Na+-H+ exchange resulting in an increase in intracellular pH and Na+ uptake concomitant with an increase in receptor-mediated endocytosis and a decrease in Na+ conductance and associated membrane hyperpolarization. The net result appears to be a down-regulation of the Na+ pump which together with a decrease in Na+ conductance may divert high-energy phosphate compounds from cation regulation to anabolic processes of meiosis.     

Characterization of the inward-rectifying potassium current in cat ventricular myocytes

Whole-cell membrane currents were measured in isolated cat ventricular myocytes using a suction-electrode voltage-clamp technique. An inward-rectifying current was identified that exhibited a time-dependent activation. The peak current appeared to have a linear voltage dependence at membrane potentials negative to the reversal potential. Inward current was sensitive to K channel blockers. In addition, varying the extracellular K+ concentration caused changes in the reversal potential and slope conductance expected for a K+ current. The voltage dependence of the chord conductance exhibited a sigmoidal relationship, increasing at more negative membrane potentials. Increasing the extracellular K+ concentration increased the maximal level of conductance and caused a shift in the relationship that was directly proportional to the change in reversal potential. Activation of the current followed a monoexponential time course, and the time constant of activation exhibited a monoexponential dependence on membrane potential. Increasing the extracellular K+ concentration caused a shift of this relationship that was directly proportional to the change in reversal potential. Inactivation of inward current became evident at more negative potentials, resulting in a negative slope region of the steady state current-voltage relationship between -140 and -180 mV. Steady state inactivation exhibited a sigmoidal voltage dependence, and recovery from inactivation followed a monoexponential time course. Removing extracellular Na+ caused a decrease in the slope of the steady state current-voltage relationship at potentials negative to -140 mV, as well as a decrease of the conductance of inward current. It was concluded that this current was IK1, the inward-rectifying K+ current found in multicellular cardiac preparations. The K+ and voltage sensitivity of IK1 activation resembled that found for the inward-rectifying K+ currents in frog skeletal muscle and various egg cell preparations. Inactivation of IK1 in isolated ventricular myocytes was viewed as being the result of two processes: the first involves a voltage-dependent change in conductance; the second involves depletion of K+ from extracellular spaces. The voltage-dependent component of inactivation was associated with the presence of extracellular Na+.     

Ionic basis of the receptor potential in primary endings of mammalian muscle spindles

The effect of changing the ionic composition of bathing fluid on the receptor potential of primary endings has been examined in isolated mammalian spindles whose capsule was removed in the sensory region. After impulse activity is blocked by tetrodotoxin, ramp-and-hold stretch evokes a characteristic pattern of potential change consisting of a greater dynamic depolarization during the ramp phase and a smaller static depolarization during the hold phase. After a high-velocity ramp there is a transient post-dynamic undershoot to below the static level. On release from hold stretch, the potential shows a postrelease undershoot relative to base line. The depolarization produced by stretch is rapidly decreased by the removal of Na+ and Ca2+. Addition of normal Ca2+ partly restores the response. Stretch appears to increase the conductance to Na+ and Ca2+ in the sensory terminals. The postdynamic undershoot is diminished by raising external K+ and blocked by tetraethylammonium (TEA). It apparently results from a voltage-dependent potassium conductance. The postrelease undershoot is decreased by raising external K+, but is not blocked by TEA. It is presumably caused by a relative increase in potassium conductance on release. Substitution of isethionate for Cl- or the addition of ouabain does not alter the postdynamic and postrelease undershoots.     

Palytoxin induces an increase in the cation conductance of red cells

Palytoxin (PTX), isolated from the marine soft coral Palythoa tuberculosa, increases the cation conductance of human red cell membranes. In the presence of 10(-10) M PTX and 10(-5) M DIDS, the membrane potential approximates the equilibrium potential for Na+ or K+ rather than Cl-. Even in the absence of DIDS, the Na+ and K+ conductances were greater than the Cl- conductance. The selectivity of the PTX-induced cation conductance is K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ much greater than choline+ greater than TEA+ much greater than Mg2+. Measurements of K+ efflux revealed two apparent sites for activation by PTX, one with a Kal of 0.05 nM and a maximum flux, nu max1, of 1.4 mol/liter of cells per h and another with a Ka2 of 98 nM and a nu max2 of 24 mol/liter of cells per h. These effects of PTX are completely blocked by external ouabain (300 microM) and prevented by internal vanadate (100 microM). When the PTX channels are open, the Na,K pumps do not catalyze ATP hydrolysis. Upon thorough washout of cells exposed to about five molecules of PTX/pump, the Na,K pump of these cells operates normally. Blockage of the positively charged NH2 terminus of PTX with a p-bromobenzoyl group reduces the potency of the compound to induce Na and K fluxes by at least a factor of 100, and to compete with the binding of [3H]ouabain by at least a factor of 10. These data are consistent with the conclusion that PTX binds reversibly to the Na,K pumps in the red cell membrane and opens a (10-pS) channel equally permeable to Na and K at or near each pump site.     

Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes

A purified (Na+ + K+)-ATPase large subunit obtained from microsomes by water-alcohol extraction was incorporated into a bilayer lipid membrane. The protein formed in the membrane conductance channels which were sensitive to ouabain and selective for monovalent cations. ATP activated these channels in the presence of sodium and potassium ions. When sodium ions were eliminated ATP did not change the conductance of the modified membrane whereas p-nitrophenyl phosphate increased it. The (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membrane possessed an ATPase activity. The presence of a potential on the membrane was a necessary condition for the enzyme incorporated into a bilayer lipid membrane to show high ATPase activity. Increasing the potential above 100 mV resulted in the closing of conductance channels.     

Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma

Membrane potentials and conductances, and intracellular ionic activities were studied in isolated perfused collecting tubules of K+-adapted Amphiuma. Intracellular Na+ (aNai) and K+ (aKi) activities were measured, using liquid ion-exchanger double-barreled microelectrodes. Apical and basolateral membrane conductances were estimated by cable analysis. The effects of inhibition of the apical conductance by amiloride (10(-5) M) and of inhibition of the basolateral Na-K pump by either a low K+ (0.1 mM) bath or by ouabain (10(-4) M) were studied. Under control conditions, aNai was 8.4 +/- 1.9 mM and aKi 56 +/- 3 mM. With luminal amiloride, aNai decreased to 2.2 +/- 0.4 mM and aKi increased to 66 +/- 3 mM. Ouabain produced an increase of aNai to 44 +/- 4 mM, and a decrease of aKi to 22 +/- 6, and similar changes were observed when the tubule was exposed to a low K+ bath solution. During pump inhibition, there was a progressive decrease of the K+-selective basolateral membrane conductance and of the Na+ permeability of the apical membrane. A similar inhibition of both membrane conductances was observed after pump inhibition by low K+ solution. Upon reintroduction of K+, a basolateral membrane hyperpolarization of -23 +/- 4 mV was observed, indicating an immediate reactivation of the electrogenic Na-K pump. However, the recovery of the membrane conductances occurred over a slower time course. These data imply that both membrane conductances are regulated according to the intracellular ionic composition, but that the basolateral K+ conductance is not directly linked to the pump activity.     

Ba2+ unmasks K+ modulation of the Na+-K+ pump in the frog retinal pigment epithelium

This paper presents electrophysiological evidence that small changes in [K+]o modulate the activity of the Na+-K+ pump on the apical membrane of the frog retinal pigment epithelium (RPE). This membrane also has a large relative K+ conductance so that lowering [K+]o hyperpolarizes it and therefore increases the transepithelial potential (TEP). Ba2+, a K+ channel blocker, eliminated these normal K+-evoked responses; in their place, lowering [K+]o evoked an apical depolarization and TEP decrease that were blocked by apical ouabain or strophanthidin. These data indicate that Ba2+ blocked the major K+ conductance(s) of the RPE apical membrane and unmasked a slowing of the normally hyperpolarizing electrogenic Na+-K+ pump caused by lowering [K+]o. Evidence is also presented that [K+]o modulates the pump in the isolated RPE under physiological conditions (i.e., without Ba2+). In the intact retina, light decreases subretinal [K+]o and produces the vitreal-positive c-wave of the electroretinogram (ERG) that originates primarily in the RPE from a hyperpolarization of the apical membrane and TEP increase. When Ba2+ was present in the retinal perfusate, the apical membrane depolarized in response to light and the TEP decreased so that the ERG c-wave inverted. The retinal component of the c-wave, slow PIII, was abolished by Ba2+. The effects of Ba2+ were completely reversible. We conclude that Ba2+ unmasks a slowing of the RPE Na+-K+ pump by the light-evoked decrease in [K+]o. Such a response would reduce the amplitude of the normal ERG c-wave.     

Direct measurement of the membrane potential of Ehrlich ascites tumor cells: lack of effect of valinomycin and ouabain.

The membrane potential of Ehrlich ascites tumor cells and the effects of valinomycin and ouabain upon it have been determined. The membrane potential in control cells was 12.0 mV, inside negative. Neither valinomycin nor ouabain alone affected this value. However, valinomycin and ouabain in combination resulted in a slight hyperpolarization of the membrane. Concomitant determinations of cellular Na+, K+ and Cl- showed that valinomycin induced net losses of K+ and Cl- and a net gain in Na+ when compared to ouabain-inhibited cells. K+ permeability was increased by approximately 30% in the presence of valinomycin. In addition, valinomycin caused a rapid depletion of cellular ATP. Inhibition of Na/K transport by ouabain was without sparing effect on the rate of ATP depletion. Possible mechanisms for the electroneutral increase in K+ permeability induced by valinomycin are discussed.     

Barium inhibition of the collapse of the Shaker K(+) conductance in zero K(+)

In the absence of K(+) on both sides of the membrane, delivery of standard activating pulses collapses the Shaker B K(+) conductance. Prolonged depolarizations restore the ability to conduct K(+). It has been proposed that the collapse of the conductance results from the dwelling of the channels in a stable closed (noninactivated) state (, J. Physiol. (Lond.). 499:3-15). Here it is shown that 1) Ba(2+) impedes the collapse of the K(+) conductance, protecting it from both sides of the membrane; 2) external Ba(2+) protection (K(d) = 63 microM at -80 mV) decreases slightly as the holding potential (HP) is made more negative; 3) external Ba(2+) cannot restore the previously collapsed conductance; on the other hand, 4) internal Ba(2+) (and K(+)) protection markedly decreases with hyperpolarized HPs (-80 to -120 mV), and it is not dependent on the pulse potential (0 to +60 mV). Ba(2+) is an effective K(+) substitute, inhibiting the passage of the channels into the stable nonconducting (noninactivated) mode of gating.     

Correlation of the effect of Ca+2 on Na+ and K+ permeability and membrane potential of Ehrlich ascites tumor cells

The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2-induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2 mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl- in steady cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport-inhibited cells. The membrane potential of cells in Ca+2-free medium (-13.9 +/- 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2-containing medium (-23.3 +/- 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (-15.2 +/- 0.7 mV). The results of these experiments are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leading to an electrogenic hyperpolarization of the membrane.     

3H]dizocilpine binding to N-methyl-D-aspartate (NMDA) receptor is modulated by an endogenous Na+, K+-ATPase inhibitor. Comparison with ouabain

An endogenous Na+, K+-ATPase inhibitor termed endobain E has been isolated from rat brain which shares several biological properties with ouabain. This cardiac glycoside possesses neurotoxic properties attributable to Na+, K+-ATPase inhibition, which leads to NMDA receptor activation, thus supporting the concept that Na+/K+ gradient impairment has a critical impact on such receptor function. To evaluate potential direct effects of endobain E and ouabain on NMDA receptors, we assayed [3H]dizocilpine binding employing a system which excludes ionic gradient participation. Brain membranes thoroughly washed and stored as pellets ('non-resuspended' membranes) or after resuspension in sucrose ('resuspended' membranes) were employed. Membrane samples were incubated with 4 or 10 nM ligand with or without added endobain E or ouabain, in the presence of different glutamate plus glycine combinations, with or without spermidine. [3H]dizocilpine basal binding and Na+, K+- and Mg2+-ATPase activities proved very similar in 'non-resuspended' or 'resuspended' membranes. Endobain E decreased [3H]dizocilpine binding to 'resuspended' membranes in a concentration-dependent manner, attaining roughly 50% binding inhibition with the highest endobain E concentration assayed. Among tested conditions, only in 'resuspended' membranes, with 4 nM ligand and with 1x10(-8) M glutamate plus 1x10(-5) M glycine, was [3H]dizocilpine binding enhanced roughly +24% by ouabain (1 mM). After Triton X-100 membrane treatment, which drastically reduces Na+, K+-ATPase activity, the effect of ouabain on binding was lost whereas that of endobain E remained unaltered. Results indicate that not only membrane preparation but also treatment and storage are crucial to observe direct endobain E and ouabain effects on NMDA receptor, which are not attributable to changes in Na+, K+-ATPase activity or to Na+/K+ equilibrium alteration.     

Serum induces the immediate opening of Ca2+-activated channels in quiescent human fibroblasts

Application of fetal calf serum to quiescent human fibroblasts produces an immediate (3-20 s delay) increase in membrane conductance which lasts about 20-30 s. This conductance is strongly outwardly-rectifying and has a reversal potential between -45 and -10 mV. The conductance increase may also be induced by application of the Ca2+ ionophore A23187 while it does not occur when intracellular K+ is replaced by Cs+. It is concluded that this early effect of serum is due to the opening of Ca2+-activated channels. This permeability change will alter the membrane potential and thus possibly interact with other voltage-sensitive processes induced by serum growth factors.     

Multiple conductances in the large K+ channel from Chara corallina shown by a transient analysis method.

The large conductance K+ channel in the tonoplast of Chara corallina has subconductance states (substates). We describe a method that detects substates by monitoring the time derivative of channel current. Substates near to the full conductance tend to have long durations and high probabilities, while those of smaller amplitude occur with less probability and short duration. The substate pattern is similar in cell-attached, inside-out and outside-out patches over a range of temperatures. The pattern changes at high Ca2+ concentration (10 mol m-3) on the cytoplasmic face of inside-out patches. One substate at approximately 50% of the full conductance is characterized by a high frequency of transitions from the full conductance level. This midstate conductance is not a constant proportion of the full conductance but changes as a function of membrane potential difference (p.d.) showing strong inward rectification. We suggest that the channel is a single pore that can change conformation and/or charge profile to give different conductances. The mean durations of the full conductance level and the midstate decrease as the membrane p.d. becomes more negative. Programs for analysis of channel kinetics based on an half-amplitude detection criterion are shown to be unsuitable for analysis of the K+ channel.     

Coupling of Li+ distribution to the plasma membrane potential of rat cortical synaptosomes

The effect of the plasma membrane potential delta psi p on the transport rate and steady state distribution of Li+ was assessed in rat cortical synaptosomes. Up to 15 mM Li+ failed to saturate Li+ influx into polarized synaptosomes in a Na+-based medium with 3 mM external K+. Veratridine increased and tetrodotoxin, ouabain, or high external K+ decreased the rate of Li+ influx. At steady state, Li+ was concentrated about 3-fold in resting synaptosomes at 0.3 to 1 mM Li+ externally. Subsequent depolarization of the plasma membrane by veratridine or high external K+ induced an immediate release of Li+. When graded depolarizations were imposed onto the plasma membrane by varying concentrations of ouabain, veratridine, or external K+, steady state distribution of Li+ was linearly related with K+ distribution or electrochemical activity coefficients. It was concluded that uptake rate and steady state distribution of Li+ depend significantly on delta psi p. However, Li+ gradients were lower than predicted from delta psi p, suggesting that (secondary) active transport systems counteracted passive equilibration by uphill extrusion of Li+. The electrochemical potential difference delta mu Li+ maintained at a delta psi p of -72 mV was calculated to 4.2 kJ/mol of Li+. At physiological external K+, Li+ was not actively transported by the sodium pump. The ouabain sensitivity resulted from the coupling of Li+ uptake to the pump-dependent K+ diffusion potential. In low K+ and K+-free media, however, active transport of Li+ by the sodium pump contributed to total uptake. In the absence of K+, Li+ substituted for K+ in generating a delta psi p of -64 mV maximally, as calculated from TPMP+ distribution at 40 mM external Li+. Since Li+ gradients were far too low to account for a diffusion potential, it was assumed that Li+ gave rise to an electrogenic pump potential.     

Normal and anomalous potential responses due to K+ changes in bullfrog antrum

The effect of changing the K+ concentration in the bathing media was studied in the bullfrog antrum. Usually increasing K+ on the nutrient side in standard Cl- -containing and Cl- -free solutions decreased the transmucosal potential difference (nutrient became more negative) - a normal effect. Similar results were obtained on the secretory side. Moreover, for K+ changes on the nutrient side in Cl- media, a plot of magnitude of delta V vs. log [K+] was linear for [K+] greater than 20 mM with slope of 27 mV per 10-fold change in [K+]. However, after bathing the mucosa in Cl- media with zero K+ for about 20 min, elevating the nutrient [K+] to 4 mM increased the potential difference (V) by 4.8 mV in 5 min and repeating the same sequence increased V by 6.9 mV in 5 min - both anomalous effects. Beyond 20 mM K+ the response was normal. In SO2-4 media, an anomalous potential difference of about 1 mV was obtained for changes from 0 to 3 or 6 mM nutrient K+. Ouabain (1 X 10(-3) M) in the nutrient solution abolished the anomalous response in Cl- and SO2-4 media. The normal response is attributed to passive, conductance pathways and the anomalous response because of the effect of ouabain, to a (Na+ + K+)-ATPase pump on the nutrient-facing membrane in which more Na+ than K+ ions are transported per cycle.     

Effect of thallium ions on gramicidin-induced conductance of muscle fiber membranes

Conductance of single fibres from m. ileofibularis of Rana esculenta was studied in isotonic K2SO4 solution under constant current conditions using the double sucrose gap method. It was found that Tl+ (at concentrations 5, 10, and 20 mM) blocked K+ currents in the gramicidin channel. The decrease of K+ conductance caused by Tl+ was associated with the changes of the membrane potential. Both the decrease of K+ conductance and value of permeability ratio (PTl/PK) found from the membrane potential changes depended on Tl+ concentration in the bathing solution. No effect of Tl+ on the potassium channels was registered in the absence of gramicidin channels. The Tl+ block described here proves the existence of Tl+ ion binding within gramicidin channels of the muscle membrane and interactions among ions in the channels.     

Comparative study of the effects of antibodies to S-100 protein and ouabain on neurons of Helix pomatia

Ouabain, used in 5.10(-4) M concentration, elicits 12 +/- 5 mV (15 experiments) depolarization of membrane of snail Helix neurons. In 80% of experiments depolarization is not accompanied by changes in membrane conductance, in 20% of experiments the decrease of the membrane conductance is observed. Application of the antibodies to S-100 protein (their concentration in the micropipette being 0.05 mg/ml) induces similar effects. The effects of ouabain and antibodies to S-100 protein are not additive and the main difference in their action lies in the ability of the cell to recover the resting potential of the membrane in the solution containing ouabain.     

Effect of ouabain on mechanical and electrical properties of rat and guinea pig hearts at 180 C.

The effects of ouabain 10(-6) M on rat and guinea pig hearts have been studied at 18 degrees C, in order to reduce almost fully both the Na+, K+-dependent ATPase activity and the ouabain induced inhibition of this enzyme. In isolated guinea pig hearts the positive inotropic response to ouabain obtained at 32 degrees C disappeared at 18 degrees C. On the contrary, the contractile strength of rat hearts was slightly reduced by ouabain and in the same manner at both temperatures. Current and voltage clamp experiments carried out at 18 degrees C in ventricular fibres revealed that ouabain 10(-6) M decreased both the action potential overshoot and the fast sodium current in rat and guinea pig, by reduction of the membrane sodium conductance. Ouabain did not change the calcium current in guinea pig preparations, whereas in rat heart muscle this current was reduced. The effects of ouabain on both the action potential plateau and outward repolarizing current indicated some inconsistencies from preparation to preparation and cannot therefore be considered as significant. The persistence of the ouabain induced alterations of g Na (in rat and guinea pig) and calcium current (in rat) at 18 degrees C supports the hypothesis of two ouabain cell receptors in heart muscle.     

Effect of the medium composition on the resting membrane potential in the earthworm longitudinal somatic muscle fibers

Norepinephrine or increased extracellular K+ hyperpolarize the membrane of the earthworm somatic muscle fibre, whereas removal of Cl- from external solution or a hypotonic solution depolarize the membrane. The dependence of the membrane resting potential on the extracellular K+ is quite characteristic against the background of ouabain action. A preliminary membrane depolarisation by ouabain eliminates the above effects on the membrane resting potential. The data obtained suggest that the ouabain-sensitive active ion pump directly contributes to the membrane resting potential value. This hypothesis is discussed with respect to existence of active Cl- transport combined with Na+, K(+)-pump which presumably takes part in the intracellular osmotic pressure regulation in the earthworm somatic muscle.