Modulation of proton transfer in the water wire of dioxolane-linked gramicidin channels by lipid membranes.

Proton conductance (g(H)) in single SS stereoisomers of dioxolane-linked gramicidin A (gA) channels were measured in different phospholipid bilayers at different HCl concentrations. In particular, measurements were obtained in bilayers made of 1,2-diphytanoyl 3-phosphocholine (DiPhPC) or its ethylated derivative 1,2-diphytanoyl 3-ethyl-phosphocholine (et-DiPhPC,). The difference between these phospholipids is that in et-DiPhPC one of the phosphate oxygens is covalently linked to an ethyl group and cannot be protonated. In relatively dilute acid solutions, g(H) in DiPhPC is significantly higher than in et-DiPhPC. At high acid concentrations, g(H) is the same in both diphytanoyl bilayers. Such differences in g(H) can be accounted for by surface charge effects at the membrane/solution interfaces. In the linear portion of the log g(H)-log [H] relationship, g(H) values in diphytanoyl bilayers were significantly larger (approximately 10-fold) than in neutral glyceryl monooleate (GMO) membranes. The slopes of the linear log-log relationships between g(H) and [H] in diphytanoyl and GMO bilayers are essentially the same (approximately 0.76). This slope is significantly lower than the slope of the log-log plot of proton conductivity versus proton concentration in aqueous solutions (approximately 1.00). Because the chemical composition of the membrane-channel/solution interface is strikingly different in GMO and diphytanoyl bilayers, the reduced slope in g(H)-[HCl] relationships may be a characteristic of proton transfer in the water wire inside the SS channel. Values of g(H) in diphytanoyl bilayers were also significantly larger than in membranes made of the more common biological phospholipids 1-palmitoyl 2-oleoyl phosphocholine (POPC) or 1-palmitoyl 2-oleoyl phosphoethanolamine (POPE). These differences, however, cannot be accounted for by different surface charge effects or by different internal dipole potentials. On the other hand, maximum g(H) measured in the SS channel does not depend on the composition of the bilayer and is determined essentially by the reduced mobility of protons in concentrated acid solutions. Finally, no experimental evidence was found in support of a lateral proton movement at the phospholipid/solution interface contributing to g(H) in single SS channels. Protein-lipid interactions are likely to modulate g(H) in the SS channel.

Covalently linked gramicidin channels: effects of linker hydrophobicity and alkaline metals on different stereoisomers.

The direct role of the dioxolane group on the gating and single-channel conductance of different stereoisomers of the dioxolane-linked gramicidin A (gA) channels reconstituted in planar lipid bilayers was investigated. Four different covalently linked gA dimers were synthesized. In two of them, the linker was the conventional dioxolane described previously (SS and RR channels). Two gAs were covalently linked with a novel modified dioxolane group containing a retinal attachment (ret-SS and ret-RR gA dimers). These proteins also formed ion channels in lipid bilayers and were selective for monovalent cations. The presence of the bulky and hydrophobic retinal group immobilizes the dioxolane linker in the bilayer core preventing its rotation into the hydrophilic lumen of the pore. In 1 M HCl the gating kinetics of the SS or RR dimers were indistinguishable from their retinal counterparts; the dwell-time distributions of the open and closed states in the SS and ret-SS were basically the same. In particular, the inactivation of the RR was not prevented by the presence of the retinal group. It is concluded that neither the fast closing events in the SS or RR dimers nor the inactivation of the RR are likely to be a functional consequence of the flipping of the dioxolane inside the pore of the channel. On the other hand, the inactivation of the RR dimer was entirely eliminated when alkaline metals (Cs(+) or K(+)) were the permeating cations in the channel. In fact, the open state of the RR channel became extremely stable, and the gating characteristics of both the SS and RR channels were different from what was seen before with permeating protons. As in HCl, the presence of a retinal in the dioxolane linker did not affect the gating behavior of the SS and RR in Cs(+)- or K(+)-containing solutions. Alternative hypotheses concerning the gating of linked gA dimers are discussed.

Gating and permeation in ion channels formed by gramicidin A and its dioxolane-linked dimer in Na(+) and Cs(+) solutions.

The association of two gramicidin A (gA) peptides via H-bonds in lipid bilayers causes the formation of an ion channel that is selective for monovalent cations only. In this study, two gAs were covalently linked with a dioxolane group (SS dimer). Some functional properties of natural gA channels were compared to that synthetic dimer in Na(+)- or Cs(+)-containing solutions. The SS dimer remained in the open configuration most of the time, while natural gA channels had a relatively brief mean open time. Single channel conductances to Na(+) (g(Na)) or Cs(+) (g(Cs)) in the SS dimer were smaller than in natural gA. However, g(Na) was considerably more attenuated than g(Cs). This probably results from a tight solvation of Na(+) by the dioxolane linker in the SS channel. In Cs(+) solutions, the SS had frequent closures. By contrast, in Na(+) solutions the synthetic dimer remained essentially in the open state. The mean open times of SS channels in different solutions (T(open, Na) > T(open,Cs) > T(open,H)) were inversely proportional to the single channel conductances (g(H) > g(Cs) > g(Na)). This suggests that ion occupancy inside the pore stabilizes the open configuration of the gA dimer. The mean closed time of the SS dimer was longer in Cs(+) than in H(+) solutions. Possible mechanisms for these effects are discussed.

Proton mobilities in water and in different stereoisomers of covalently linked gramicidin A channels.

Proton conductivities in bulk solution (lambda(H)) and single-channel proton conductances (g(H)) in two different stereoisomers of the dioxolane-linked gramicidin A channel (the SS and RR dimers) were measured in a wide range of bulk proton concentrations ([H], 0.1-8000 mM). Proton mobilities (micro(H)) in water as well as in the SS and RR dimers were calculated from the conductivity data. In the concentration range of 0.1-2000 mM, a straight line with a slope of 0.75 describes the log (g(H))-log ([H]) relationship in the SS dimer. At [H] > 2000 mM, saturation is followed by a decline in g(H). The g(H)-[H] relationship in the SS dimer is qualitatively similar to the [H] dependence of lambda(H). However, the slope of the straight line in the log(lambda(H))-log([H]) plot is 0.96, indicating that the rate-limiting step for proton conduction through the SS dimer is not the diffusion of protons in bulk solution. The significant difference between the slopes of those linear relationships accounts for the faster decline of micro(H) as a function of [H] in the SS dimer in relation to bulk solution. In the high range of [H], saturation and decline of g(H) in the SS dimer can be accounted for by the significant decrease of micro(H) in bulk solution. At any given [H], g(H) in the RR dimer is significantly smaller than in the SS. Moreover, the g(H)-[H] relationship in the RR stereoisomer is qualitatively different from that in the SS. Between 1 and 50 mM [H], g(H) can be fitted with an adsorption isotherm, suggesting the presence of a proton-binding site inside the pore (pK(a) approximately 2), which limits proton exit from the channel. At 100 mM < [H] < 3000 mM, g(H) increases linearly with [H]. The distinctive shape of the g(H)-[H] relationship in the RR dimer suggests that the channel can be occupied simultaneously by more than one proton. At higher [H], the saturation and decline of g(H) in the RR dimer reflect the properties of micro(H) in bulk solution. In the entire range of [H], protons seem to cross the SS and RR channels via a Grotthuss-like mechanism. The rate-limiting step for proton transfer in the SS dimer is probably the membrane-channel/bulk solution interface. It is also proposed that the smaller g(H) in the RR dimer is the consequence of a different organization and dynamics of the H-bonded network of water molecules inside the pore of the channel, resulting in a slower proton transfer and multiple pore occupancy by protons.

The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels.

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.

Attenuation of proton currents by methanol in a dioxolane-linked gramicidin A channel in different lipid bilayers.

The mobility of protons in a dioxolane-linked gramicidin A channel (D1) is comparable to the mobility of protons in aqueous solutions (Cukierman, S., E. P. Quigley, and D. S. Crumrine. 1997. Biophys. J. 73:2489-2502). Aliphatic alcohols decrease the mobility of H+ in aqueous solutions. In this study, the effects of methanol on proton conduction through D1 channels were investigated in different lipid bilayers and at different HCl concentrations. Methanol attenuated H+ currents in a voltage-independent manner. Attenuation of proton currents was also independent of H+ concentrations in solution. In phospholipid bilayers, methanol decreased the single channel conductance to protons without affecting the binding affinity of protons to bilayers. In glycerylmonooleate membranes, the attenuation of single channel proton conductances qualitatively resembled the decrease of conductivities of HCl solutions by methanol. However, in both types of lipid bilayers, single channel proton conductances through D1 channels were considerably more attenuated than the conductivities of different HCl solutions. This suggests that methanol modulates single proton currents through D1 channels. It is proposed that, on average, one methanol molecule binds to a D1 channel, and attenuates H+ conductance. The Gibbs free energy of this process (DeltaG0) is approximately 1.2 kcal/mol, which is comparable to the free energy of decrease of HCl conductivity in methanol solutions (1.6 kcal/mol). Apolar substances like urea and glucose that do not transport protons in HCl solutions and do not permeate D1 channels decreased solution conductivity and single channel conductance by a considerably larger proportion than methanol. Cs+ currents through D1 channels were considerably less (fivefold) attenuated by methanol than proton currents. It is proposed that methanol partitions inside the pore of gramicidin channels and delays the transfer of protons between water and methanol molecules, causing a significant attenuation of the single channel proton conductance. Gramicidin channels offer an interesting experimental model to study proton hopping along a single chain of water molecules interrupted by a single methanol molecule.

Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers.

Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA dimers. g(max) and K(D), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its dimer. In GMO bilayers, however, proton affinities of gA and the dioxolane-dimer were significantly lower (K(D) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and 2150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers.

Direct modulation of Na+ currents by protein kinase C activators in mouse neuroblastoma cells.

We investigated the effects of different protein kinase C (PKC) activators on Na+ currents using the conventional whole-cell and the inside-out macropatch voltage-clamp techniques in mouse neuroblastoma cells (N1E-115). Two different categories of PKC activators were investigated: the cis-unsaturated fatty acids (CUFAs): oleic (cis-9-octadecenoic), linoleic (cis-9-12-octadecadienoic), and linolenic acid (cis-9-12-15-octadecatrienoic), and, the diacylglycerol (DAG) derivative 1-2-dioctanoyl-sn-glycerol (DOG). These substances caused the following alterations on Na+ currents: (i) Na+ currents were attenuated as a function of voltage. While DOG attenuated both inward and outward Na+ currents in a monotonic and continuous voltage-dependent manner, CUFAs preferentially attenuated inward currents; (ii) the steady-state activation curve of Na+ currents shifted to more depolarized voltages; (iii) opposite to the activation curve, the steady-state inactivation curve of Na+ channels (h curve) shifted to more hyperpolarized voltages; (iv) the time course of inactivation development was accelerated by PKC activators, while the recovery from inactivation was not affected; (v) substances that inhibit different metabolic pathways (PKC activation, cyclooxygenase, lipooxygenase, and P-450 pathways) did not prevent the effects of PKC activators on Na+ currents. One fully saturated fatty acid (octadecanoic acid), a trans-unsaturated fatty acid (trans-9-octadecenoic), and different phorbol esters did not affect Na+ currents; (vi) effects of different PKC activators on Na+ currents were completely reversible. These observations suggest that PKC activators might interact with Na+ channels directly. These direct effects must be taken into consideration in evaluating the overall effect of PKC activation on Na+ channels. Moreover, it is likely that this direct interaction could account, at least in part, for the diversity of effects of PKC activators on Na+ channels.

Diacylglycerol-induced activation of protein kinase C attenuates Na+ currents by enhancing inactivation from the closed state.

The causes of attenuation of Na+ currents by diacylglycerol (DAG)-induced protein kinase C (PKC) activation in mouse neuroblastoma N1E-115 cells were investigated using the cell-attached patch, and the perforated-patch (nystatin based) whole-cell voltage-clamp techniques. Activation of PKC by DAG attenuated Na+ currents. Attenuation occurred in the absence of significant changes in the time-course of Na+ currents. However, the steady-state inactivation curve of these currents shifted to more negative voltages by approximately 20 mV. Here we demonstrate that the time-course of inactivation is accelerated by treatment with DAG-like substances in a voltage-dependent manner (time constant of inactivation decreased by 2- and 3.6-fold at -60, and -30 mV, respectively). In cell-attached patches, treatment with DAG compounds increased the percentage of current traces showing no single Na+ channel openings in response to depolarizing voltage-clamp pulses. Moreover, the average of current traces containing single Na+ channel openings was essentially the same in control conditions and after treatment with DAG compounds. Removal of Na+ channel inactivation by the alkaloid batrachotoxin prevented the attenuation of Na+ currents by PKC activation via DAGs. Taken together, these data strongly suggest that PKC-induced attenuation of Na+ currents is linked to an enhancement of Na+ channel inactivation. This attenuation is caused by an increase in the number of Na+ channels inactivating directly from the closed state(s). This inactivation pathway represents a simple and efficient physiological mechanism by which PKC activation might modulate the electrical activity of excitable cells.

Multiple effects of protein kinase C activators on Na+ currents in mouse neuroblastoma cells.

The effects of externally applied different protein kinase C (PKC) activators on Na+ currents in mouse neuroblastoma cells were studied using the perforated-patch (nystatin-based) whole cell voltage clamp technique. Two diacylglycerol-like compounds, OAG (1-oleoyl-2-acetyl-sn-glycerol), and DOG (1-2-dioctanoyl-rac-glycerol) attenuated Na+ currents without affecting the time course of activation or inactivation. The reduction in Na+ current amplitude caused by OAG or DOG was dependent on membrane potential, being more intense at positive voltages. The steady-state activation curve was also unaffected by these substances. However, both OAG and DOG shifted the steady-state inactivation curve of Na+ currents to more hyperpolarized voltages. Surprisingly, phorbol esters did not affect Na+ currents. Cis-unsaturated fatty acids (linoleic, linolenic, and arachidonic) attenuated Na+ currents without modifying the steady-state activation. As with DOG and OAG, cis-unsaturated fatty acids also shifted the steady-state inactivation curve to more negative voltages. Interestingly, inward currents were more effectively attenuated by cis-fatty acids than outward currents. Oleic acid, also a cis-unsaturated fatty acid, enhanced Na+ currents. This enhancement was not accompanied by changes in kinetic or steady-state properties of currents. Enhancement of Na+ currents caused by oleate was voltage dependent, being stronger at negative voltages. The inhibitory or stimulatory effects caused by all PKC activators on Na+ currents were completely prevented by pretreating cells with PKC inhibitors (calphostin C, H7, staurosporine or polymyxin B). By themselves, PKC inhibitors did not affect membrane currents. Trans-unsaturated or saturated fatty acids, which do not activate PKC's, did not modify Na+ currents. Taken together, the experimental results suggest that PKC activation modulates the behavior of Na+ channels by at least three distinct mechanisms. Because qualitatively different results were obtained with different PKC activators, it is not clear how Na+ currents would respond to activation of PKC under physiological conditions.

Effects of extracellular sodium, quinidine, and ryanodine on slow response excitability in rabbit atrial trabeculae.

1. We investigated Na(+)-Ca2+ exchange and the involvement of the sarcoplasmic reticulum in frequency-dependent slow response excitability enhancement in rabbit atrial trabeculae. 2. Slow responses were induced in a modified Tyrode solution containing high K+ and Ba2+ and conventional electrophysiological techniques were used for stimulating and recording membrane potentials. 3. Under these conditions, the frequency-dependence of slow response excitability can be demonstrated with excitability enhancement as stimulation frequency is increased (0.25 to 1.0 Hz). 4. The frequency-dependent excitability enhancement depends on external Na+, increasing in high-[Na+]o (173.8 mM) and decreasing in low-[Na+]o (103.8 mM) media. 5. Quinidine (10 microM) and ryanodine (10 microM) decrease frequency-dependent slow response excitability enhancement. 6. These results indicate that the Na(+)-Ca2+ exchange might have an important role in frequency-dependent excitability enhancement of slow responses. Moreover, we suggest that the control of internal Ca2+ by the sarcoplasmic reticulum might have an additional role in regulating the excitability enhancement process in depolarized atrial trabeculae.

Effects of extracellular sodium, quinidine, and ryanodine on slow response excitability in rabbit atrial trabeculae

1. We investigated Na(+)-Ca2+ exchange and the involvement of the sarcoplasmic reticulum in frequency-dependent slow response excitability enhancement in rabbit atrial trabeculae. 2. Slow responses were induced in a modified Tyrode solution containing high K+ and Ba2+ and conventional electrophysiological techniques were used for stimulating and recording membrane potentials. 3. Under these conditions, the frequency-dependence of slow response excitability can be demonstrated with excitability enhancement as stimulation frequency is increased (0.25 to 1.0 Hz). 4. The frequency-dependent excitability enhancement depends on external Na+, increasing in high-[Na+]o (173.8 mM) and decreasing in low-[Na+]o (103.8 mM) media. 5. Quinidine (10 microM) and ryanodine (10 microM) decrease frequency-dependent slow response excitability enhancement. 6. These results indicate that the Na(+)-Ca2+ exchange might have an important role in frequency-dependent excitability enhancement of slow responses. Moreover, we suggest that the control of internal Ca2+ by the sarcoplasmic reticulum might have an additional role in regulating the excitability enhancement process in depolarized atrial trabeculae

Barium modulates the gating of batrachotoxin-treated Na+ channels in high ionic strength solutions.

Batrachotoxin-activated rat brain Na+ channels were reconstituted in neutral planar phospholipid bilayers in high ionic strength solutions (3 M NaCl). Under these conditions, diffuse surface charges present on the channel protein are screened. Nevertheless, the addition of extracellular and/or intracellular Ba2+ caused the following alterations in the gating of Na+ channels: (a) external (or internal) Ba2+ caused a depolarizing (or hyperpolarizing) voltage shift in the gating curve (open probability versus membrane potential curve) of the channels; (b) In the concentration range of 10-120 mM, extracellular Ba2+ caused a larger voltage shift in the gating curve of Na+ channels than intracellular Ba2+; (c) voltage shifts of the gating curve of Na+ channels as a function of external or internal Ba2+ were fitted with a simple binding isotherm with the following parameters: for internal Ba2+, delta V0.5,max (maximum voltage shift) = -11.5 mV, KD = 64.7 mM; for external Ba2+, delta V0.5,max = 13.5 mV, KD = 25.8 mM; (d) the change in the open probability of the channel caused by extracellular or intracellular Ba2+ is a consequence of alterations in both the opening and closing rate constants. Extracellular and intracellular divalent cations can modify the gating kinetics of Na+ channels by a specific modulatory effect that is independent of diffuse surface potentials. External or internal divalent cations probably bind to specific charges on the Na+ channel glycoprotein that modulate channel gating.

K-current mediation of prolactin-induced proliferation of malignant (Nb2) lymphocytes.

The effects of different concentrations of various K-current blockers on prolactin-induced proliferation and membrane K-currents in malignant lymphocytes (Nb2 cells) were investigated. Membrane currents were measured with the whole cell patch-clamp technique, and lymphocyte density was quantified by both spectrophotometric and conventional methods. K-current blockers tested (quinidine, 4-aminopyridine, barium, and tetraethylammonium) exhibited similar rank order potency for K-current block and inhibition of prolactin-induced proliferation of malignant lymphocytes. Because Nb2 cells proliferate independently of a transmembrane Ca-influx, these results suggest that K-currents per se rather than K-current modulation of Ca-influx is an essential event for lymphocyte proliferation.

Characterization of K+ currents in rat malignant lymphocytes (Nb2 cells).

Membrane K+ currents of malignant lymphocytes (Nb2 cells) were studied with the whole-cell patch-clamp method. Upon depolarization, K+ currents activate with a delay and follow a sigmoid time course, resembling other delayed rectifier K+ currents present in nerve and muscle cells. The activation time constant of these currents is voltage dependent, increasing from 1 msec at +90 mV to approximately 37 msec at -30 mV. The fractional number of open channels has a sigmoid voltage dependence with a midpoint near -25 mV. Deactivation of K+ currents in Nb2 cells is voltage dependent and follows a simple exponential time course. Time constant of this process increases from 5 msec at -115 mV to almost 80 msec at -40 mV. The relative permeability of K+ channels to different monovalent cations follows the sequence: K+ (1) greater than Rb+ (0.75) greater than NH4+ (0.11) greater than Cs+ (0.07) greater than Na+ (0.05). Inactivation of K+ currents is a biexponential process with time constants of approximately 600 and 7,000 msec. Inactivation of K+ currents in Nb2 cells is not a voltage-dependent process. The steady-state inactivation curve of K+ currents has a midpoint near -40 mV. Following a 500-msec voltage pulse, inactivation of K+ currents recovers with a simple exponential process with a time constant of 9 sec. Short duration (approximately 50 msec) voltage-clamp pulses do not induce significant inactivation of these currents. K+ currents in malignant lymphocytes do not display the phenomenon of cumulative inactivation as described for other delayed rectifier-type K+ channels. Application of a train of voltage pulses to positive potentials at different frequencies induces a moderate decrease in peak outward currents. The use of substances (N-bromoacetamide, trypsin, chloramine-T, and papain) that remove the inactivation of Na+ and K+ currents in other cells are not effective in removing the inactivation of K+ currents present in this lymphoma cell line. Significant differences were found between the characteristics of K+ currents in this malignant cell line and those present in normal lymphocytes. Possible physiological implications for these differences and for the role of K+ currents in the proliferation of normal and malignant lymphocytes are discussed.

Effects of internal divalent cations on the gating of rat brain Na+ channels reconstituted in planar lipid bilayers.

The effects of different intracellular divalent cations on the gating of single batrachotoxin-activated Na+ channels were investigated in planar lipid bilayers. Intracellular divalent cations increased the open probability (Po) of Na+ channels; the gating curve [Po versus membrane potential (Vm) relationship] shifted to more negative potentials. The relative ability of different intracellular divalent cations in shifting the gating curve decreased in the sequence: Mg2+, Ca2+, Ba2+, Sr2+. The cations Ca2+, Ba2+, and Sr2+ induced a larger voltage shift when applied to the extracellular than to the intracellular side of the Na+ channel, whereas, Mg2+ induced the same voltage shift from both sides. The increase in Po induced by intracellular divalent cations was the result of a simultaneous decrease in the closing rate and increase in the opening rate constant, however, the effect of intracellular divalent cations on the closing rate was larger than on the opening rate. These results suggest that there are both differences in surface charge densities between the intracellular and extracellular surfaces of the Na+ channel and differences in chemical affinities of those charges for different divalent cations. The effects of internal divalent cations on Na+ channel gating cannot be explained solely by surface charge reduction, which predicts that the opening and closing rates should be affected equally, but rather are consistent with a mechanism that involves screening and binding of surface charges present on the channel, plus a specific modulatory effect that accounts for the preferential effect of intracellular divalent cations on the closing rate constant.

Inactivation modifiers of Na+ currents and the gating of rat brain Na+ channels in planar lipid membranes.

Rat brain Na+ channels whose inactivation process had been removed either by batrachotoxin (BTX) or veratridine (VT) were reconstituted into planar lipid membranes. The voltage dependence of the open probability (Po) of the channel, of the opening and closing rate constants, and the conductance and relative permeability for Na+ and K+ were studied in voltage-clamp conditions in the presence of agents known to modify the inactivation of Na+ currents. In relation to alkaloids (BTX, VT, and aconitine), it was found that once a Na+ channel was modified by BTX or VT, the addition of another alkaloid did not change further the gating and permeation properties of the channel over a period of about 1 h. Once the inactivation process of the channels is removed by BTX, the addition of a proteolytic enzyme (trypsin) or an halogenated compound (chloramine-T, CT) induced profound and specific modifications on the opening and closing events of Na+ channels: (1) the voltage dependence of the channel Po shifted to more hyperpolarized potentials; (2) this voltage shift can be explained by equal hyperpolarizing voltage shifts of the opening and closing rate constants of the channel; (3) although the gating properties of the channel were modified by these compounds, the permeation properties of the channel, as evaluated by the conductance and the selectivity to Na+ and K+ ions, were unaltered; (4) trypsin and CT were active only in the intracellular side of the channel and were irreversible within the time course of the experiments, suggesting covalent modifications of the channel. Inactivation modifiers also affected the gating of toxin-activated single Na+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.

Reconstitution of single calcium channels from secretory granules of rat adenohypophysis in planar lipid membranes.

Free cytosolic Ca2+ ([Ca2+]i) has been demonstrated to play a crucial role in the prolactin secretory pathway. It regulates events as apparently diverse as prolactin gene transcription and the fusion of secretory granules with the plasma membrane. It is therefore important to understand the mechanisms which regulate the level of [Ca2+]i. Because prolactin secretory granule membranes have been shown to contain large amounts of Ca2+ and there is evidence that this calcium can be released independently of the granule hormone content, we have investigated the possibility that prolactin secretory granule membranes contain Ca2+ channels. When purified prolactin secretory granules were fused with an artificial phospholipid bilayer, we found a Ca2+ channel with a linear current-voltage relationship (conductance -45 pS) in symmetrical 50 mM CaCl2 solutions. Said channel had an open probability that was weakly dependent on the transmembrane potential, and a very good selectivity for calcium over chloride ions. The channel opened and closed very rapidly, when the majority of events lasting well below 25 ms. This channel could be important for the provision of high [Ca2+]i levels necessary for granule-plasma membrane fusion and could also be involved in the modulation of Ca2+ fluxes across the plasma membrane after the exocytotic release of prolactin.