Interaction of Ba2+ with the pores of the cloned inward rectifier K+ channels Kir2.1 expressed in Xenopus oocytes.

Interactions of Ba2+ with K+ and molecules contributing to inward rectification were studied in the cloned inward rectifier K+ channels, Kir2.1. Extracellular Ba2+ blocked Kir2.1 channels with first-order kinetics in a Vm-dependent manner. At Vm more negative than -120 mV, the Kd-Vm relationship became less steep and the dissociation rate constants were larger, suggesting Ba2+ dissociation into the extracellular space. Both depolarization and increasing [K+]i accelerated the recovery from extracellular Ba2+ blockade. Intracellular K+ appears to relieve Ba2+ blockade by competitively slowing the Ba2+ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100 microM) reduced, whereas 1 mM [Mg2+]i only slightly reduced, the ability of intracellular K+ to repulse Ba2+ from the channel pore. Intracellular Ba2+ also blocked outward IKir2.1 in a voltage-dependent fashion. At Vm >/= +40 mV, where intrinsic inactivation is prominent, intracellular Ba2+ accelerated the inactivation rate of the outward IKir2.1 in a Vm-independent manner, suggesting interaction of Ba2+ with the intrinsic gate of Kir2.1 channels.     

K+ channels of stomatal guard cells. Characteristics of the inward rectifier and its control by pH

Intracellular microelectrode recordings and a two-electrode voltage clamp have been used to characterize the current carried by inward rectifying K+ channels of stomatal guard cells from the broadbean, Vicia faba L. Superficially, the current displayed many features common to inward rectifiers of neuromuscular and egg cell membranes. In millimolar external K+ concentrations (Ko+), it activated on hyperpolarization with half-times of 100-200 ms, showed no evidence of time- or voltage-dependent inactivation, and deactivated rapidly (tau approximately 10 ms) on clamping to 0 mV. Steady-state conductance-voltage characteristics indicated an apparent gating charge of 1.3-1.6. Current reversal showed a Nernstian dependence on Ko+ over the range 3-30 mM, and the inward rectifier was found to be highly selective for K+ over other monovalent cations (K+ greater than Rb+ greater than Cs+ much greater than Na+). Unlike the inward rectifiers of animal membranes, the current was blocked by charybdotoxin and alpha-dendrotoxin (Kd much less than 50 nM), as well as by tetraethylammonium chloride (K1/2 = 9.1 mM); gating of the guard cell K+ current was fixed to voltages near -120 mV, independent of Ko+, and the current activated only with supramillimolar K+ outside (EK+ greater than -120 mV). Most striking, however, was inward rectifier sensitivity to [H+] with the K+ current activated reversibly by mild acid external pH. Current through the K+ inward rectifier was found to be largely independent of intracellular pH and the current reversal (equilibrium) potential was unaffected by pHo from 7.4 to 5.5. By contrast, current through the K+ outward rectifier previously characterized in these cells (1988. J. Membr. Biol. 102:235) was largely insensitive to pHo, but was blocked reversibly by acid-going intracellular pH. The action of pHo on the K+ inward rectifier could not be mimicked by extracellular Ca2+ for which changes in activation, deactivation, and conductance were consonant with an effect on surface charge ([Ca2+] less than or equal to 1 mM). Rather, extracellular pH affected activation and deactivation kinetics disproportionately, with acid-going pHo raising the K+ conductance and shifting the conductance-voltage profile positive-going along the voltage axis and into the physiological voltage range. Voltage and pH dependencies for gating were consistent with a single, titratable group (pKa approximately 7 at -200 mV) residing deep within the membrane electric field and accessible from the outside.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vasa recta pericytes express a strong inward rectifier K+ conductance

Strong inward rectifier potassium channels are expressed by some vascular smooth muscle cells and facilitate K+-induced hyperpolarization. Using whole cell patch clamp of isolated descending vasa recta (DVR), we tested whether strong inward rectifier K+ currents are present in smooth muscle and pericytes. Increasing extracellular K+ from 5 to 50 and 140 mmol/l induced inward rectifying currents. Those currents were Ba2+ sensitive and reversed at the K+ equilibrium potential imposed by the electrode and extracellular buffers. Ba2+ binding constants in symmetrical K+ varied between 0.24 and 24 micromol/l at -150 and -20 mV, respectively. Ba2+ blockade was time and voltage dependent. Extracellular Cs+ also blocked the inward currents with binding constants between 268 and 4,938 micromol/l at -150 and -50 mV, respectively. Ba2+ (30 micromol/l) and ouabain (1 mmol/l) depolarized pericytes by an average of 11 and 24 mV, respectively. Elevation of extracellular K+ from 5 to 10 mmol/l hyperpolarized pericytes by 6 mV. That hyperpolarization was reversed by Ba2+ (30 micromol/l). We conclude that strong inward rectifier K+ channels and Na+-K+-ATPase contribute to resting potential and that KIR channels can mediate K+-induced hyperpolarization of DVR pericytes.     

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

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

Diversity of K+ channels in circular smooth muscle of opossum lower esophageal sphincter

We previously demonstrated that a balance of K+ and Ca2+-activated Cl- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES). In the current studies, the contribution of major K+ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting 400 ms (-120 mV to +100 mV) with increments of 20 mV from holding potential of -80 mV ([K+]I = 150 mM, [K+]o = 2.5 mM). However, no inward rectification was observed. The outward current peaked within 50 ms and showed little or no inactivation. It was significantly decreased by bath application of nifedipine, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and iberiotoxin (IBTN). Further combination of TEA with 4-AP, nifedipine with 4-AP, and IBTN with TEA, or vice versa, blocked more than 90% of the outward current. Ca2+-sensitive single channels were recorded at asymetrical K+ gradients in cell-attached patch-clamp configurations (100.8+/-3.2 pS, n = 8). Open probability of the single channels recorded in inside-out patch-clamp configurations were greatly decreased by bath application of IBTN (100 nM) (Vh = -14.4+/-4.8 mV in control vs. 27.3+/-0.1 mV, n = 3, P < 0.05). These data suggest that large conductance Ca2+-activated K+ and delayed rectifier K+ channels contribute to the membrane potential, and thereby regulate the basal tone of opossum LES circular smooth muscle.     

Effects of external Rb+ on inward rectifier K+ channels of bovine pulmonary artery endothelial cells

Inward rectifier (IR) K+ channels of bovine pulmonary artery endothelial cells were studied using the whole-cell, cell-attached, and outside-out patch-clamp configurations. The effects of Rb+ on the voltage dependence and kinetics of IR gating were explored, with [Rb+]o + [K+]o = 160 mM. Partial substitution of Rb+ for K+ resulted in voltage-dependent reduction of inward currents, consistent with Rb+ being a weakly permeant blocker of the IR. In cells studied with a K(+)- free pipette solution, external Rb+ reduced inward IR currents to a similar extent at large negative potentials but block at more positive potentials was enhanced. In outside-out patches, the single-channel i-V relationship was approximately linear in symmetrical K+, but rectified strongly outwardly in high [Rb+]o due to a reduced conductance for inward current. The permeability of Rb+ based on reversal potential, Vrev, was 0.45 that of K+, whereas the Rb+ conductance was much lower, 0.034 that of K+, measured at Vrev-80 mV. The steady state voltage- dependence of IR gating was determined in Rb(+)-containing solutions by applying variable prepulses, followed by a test pulse to a potential at which outward current deactivation was observed. As [Rb+]o was increased, the half-activation potential, V1/2, changed less than Vrev. In high [K+]o solutions V1/2 was Vrev-6 mV, while in high [Rb+]o V1/2 was Vrev + 7 mV. This behavior contrasts with the classical parallel shift of V1/2 with Vrev in K+ solutions. Steady state IR gating was less steeply voltage-dependent in high [Rb+]o than in K+ solutions, with Boltzmann slope factors of 6.4 and 4.4 mV, respectively. Rb+ decreased (slowed) both activation and deactivation rate constants defined at V1/2, and decreased the steepness of the voltage dependence of the activation rate constant by 42%. Deactivation of IR channels in outside-out patches was also slowed by Rb+. In summary, Rb+ can replace K+ in setting the voltage-dependence of IR gating, but in doing so alters the kinetics.     

Vasoactive intestinal peptide-stimulated Cl- secretion: activation of cAMP-dependent K+ channels

Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.     

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption

Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++-sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.     

The pacemaker current in cardiac Purkinje myocytes

It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward current i(f) underlies the pacemaker potential. Because some findings are at odds with this interpretation, we used the whole cell patch clamp method to study the currents in the voltage range of diastolic depolarization in single canine Purkinje myocytes, a preparation where many confounding limitations can be avoided. In Tyrode solution ([K+]o = 5.4 mM), hyperpolarizing steps from Vh = -50 mV resulted in a time-dependent inwardly increasing current in the voltage range of diastolic depolarization. This time- dependent current (iKdd) appeared around -60 mV and reversed near EK. Small superimposed hyperpolarizing steps (5 mV) applied during the voltage clamp step showed that the slope conductance decreases during the development of this time-dependent current. Decreasing [K+]o from 5.4 to 2.7 mM shifted the reversal potential to a more negative value, near the corresponding EK. Increasing [K+]o to 10.8 mM almost abolished iKdd. Cs+ (2 mM) markedly reduced or blocked the time-dependent current at potentials positive and negative to EK. Ba2+ (4 mM) abolished the time-dependent current in its usual range of potentials and unmasked another time-dependent current (presumably i(f)) with a threshold of approximately -90 mV (> 20 mV negative to that of the time-dependent current in Tyrode solution). During more negative steps, i(f) increased in size and did not reverse. During i(f) the slope conductance measured with small (8-10 mV) superimposed clamp steps increased. High [K+]o (10.8 mM) markedly increased and Cs+ (2 mM) blocked i(f). We conclude that: (a) in the absence of Ba2+, a time-dependent current does reverse near EK and its reversal is unrelated to K+ depletion; (b) the slope conductance of that time-dependent current decreases in the absence of K+ depletion at potentials positive to EK where inactivation of iK1 is unlikely to occur. (c) Ba2+ blocks this time-dependent current and unmasks another time-dependent current (i(f)) with a more negative (> 20 mV) threshold and no reversal at more negative values; (d) Cs+ blocks both time-dependent currents recorded in the absence and presence of Ba2+. The data suggest that in the diastolic range of potentials in Purkinje myocytes there is a voltage- and time-dependent K+ current (iKdd) that can be separated from the hyperpolarization- activated inward current i(f).     

Slow membrane potential changes in skeletal muscle induced in Cl(-)-free medium.

1. Conventional microelectrode techniques were used to measure simultaneous changes in membrane potential (Vm) and conductance (Gm) induced by single electrical stimuli in muscles bathed in Cl(-)-free solution containing 40 mM of tetraethylammonium (TEA+). 2. Stimulation induced slow transient depolarizations (slow response) accompanied by increased calcium conductance, while the potassium conductance was first elevated and later reduced. 3. Stepwise elevation of [K+]0 from 2.5 to 5 or 10 mM during the slow response evoked an abrupt repolarization of 42.3 +/- 8.9 mV (n = 4; p less than 0.001), and 24.8 +/- 3.5 +/- mV (n = 5; p less than 0.001), respectively, while Gm was increased to 1.45 +/- 0.25-fold (n = 5; p less than 0.05). Neither the slow response nor K(+)-induced changes in Vm or Gm were sensitive to tetrodotoxin (3 microM), however, nifedipine (10 microM) abolised the slow response. 4. It was concluded that beyond the increase of calcium conductance, the ionic conductance of the inward rectifier K+ channel was reduced during the slow response, which could be restored by the elevation of [K+]0. The results suggest the possible contribution of these mechanisms to the electrical instability of myotonic muscles. Potential therapeutic consequences are discussed.     

Ionic currents and ion channels of lobster olfactory receptor neurons

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.     

Permeation of Na+ through a delayed rectifier K+ channel in chick dorsal root ganglion neurons

In whole-cell patch clamp recordings from chick dorsal root ganglion neurons, removal of intracellular K+ resulted in the appearance of a large, voltage-dependent inward tail current (Icat). Icat was not Ca2+ dependent and was not blocked by Cd2+, but was blocked by Ba2+. The reversal potential for Icat shifted with the Nernst potential for [Na+]. The channel responsible for Icat had a cation permeability sequence of Na+ >> Li+ >> TMA+ > NMG+ (PX/PNa = 1:0.33:0.1:0) and was impermeable to Cl-. Addition of high intracellular concentrations of K+, Cs+, or Rb+ prevented the occurrence of Icat. Inhibition of Icat by intracellular K+ was voltage dependent, with an IC50 that ranged from 3.0-8.9 mM at membrane potentials between -50 and -110 mV. This voltage- dependent shift in IC50 (e-fold per 52 mV) is consistent with a single cation binding site approximately 50% of the distance into the membrane field. Icat displayed anomolous mole fraction behavior with respect to Na+ and K+; Icat was inhibited by 5 mM extracellular K+ in the presence of 160 mM Na+ and potentiated by equimolar substitution of 80 mM K+ for Na+. The percent inhibition produced by both extracellular and intracellular K+ at 5 mM was identical. Reversal potential measurements revealed that K+ was 65-105 times more permeant than Na+ through the Icat channel. Icat exhibited the same voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in these neurons. We conclude that Icat is a Na+ current that passes through a delayed rectifier K+ channel when intracellular K+ is reduced to below 30 mM. At intracellular K+ concentrations between 1 and 30 mM, PK/PNa remained constant while the conductance at -50 mV varied from 80 to 0% of maximum. These data suggest that the high selectivity of these channels for K+ over Na+ is due to the inability of Na+ to compete with K+ for an intracellular binding site, rather than a barrier that excludes Na+ from entry into the channel or a barrier such as a selectivity filter that prevents Na+ ions from passing through the channel.     

G protein control of potassium channel activity in a mast cell line

Using the patch-clamp technique, we studied regulation of potassium channels by G protein activators in the histamine-secreting rat basophilic leukemia (RBL-2H3) cell line. These cells normally express inward rectifier K+ channels, with a macroscopic whole-cell conductance in normal Ringer ranging from 1 to 16 nS/cell. This conductance is stabilized by including ATP or GTP in the pipette solution. Intracellular dialysis with any of three different activators of G proteins (GTP gamma S, GppNHp, or AlF-4) completely inhibited the inward rectifier K+ conductance with a half-time for decline averaging approximately 300 s after "break-in" to achieve whole-cell recording. In addition, with a half-time averaging approximately 200 s, G protein activators induced the appearance of a novel time-independent outwardly rectifying K+ conductance, which reached a maximum of 1-14 nS. The induced K+ channels are distinct from inward rectifier channels, having a smaller single-channel conductance of approximately 8 pS in symmetrical 160 mM K+, and being more sensitive to block by quinidine, but less sensitive to block by Ba2+. The induced K+ channels were also highly permeable to Rb+ but not to Na+ or Cs+. The current was not activated by the second messengers Ca2+, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, or by cyclic AMP-dependent phosphorylation. Pretreatment of cells with pertussis toxin (0.1 microgram/ml for 12-13 h) prevented this current's induction both by guanine nucleotides and aluminum fluoride, but had no effect on the decrease in inward rectifier conductance. Since GTP gamma S is known to stimulate secretion from patch-clamped rat peritoneal mast cells, it is conceivable that K+ channels become inserted into the plasma membrane from secretory granules. However, total membrane capacitance remained nearly constant during appearance of the K+ channels, suggesting that secretion induced by GTP gamma S was minimal. Furthermore, pertussis toxin had no effect on secretion triggered by antigen, and triggering of secretion before electrical recording failed to induce the outward K+ current. Finally, GTP gamma S activated the K+ channel in excised inside-out patches of membrane. We conclude that two different GTP-binding proteins differentially regulate two subsets of K+ channels, causing the inward rectifier to close and a novel K+ channel to open when activated.     

Ion permeation and block of M-type and delayed rectifier potassium channels. Whole-cell recordings from bullfrog sympathetic neurons

Ion permeation and conduction were studied using whole-cell recordings of the M-current (I(M)) and delayed rectifier (IDR), two K+ currents that differ greatly in kinetics and modulation. Currents were recorded from isolated bullfrog sympathetic neurons with 88 mM [K+]i and various external cations. Selectivity for extracellular monovalent cations was assessed from permeability ratios calculated from reversal potentials and from chord conductances for inward current. PRb/PK was near 1.0 for both channels, and GRb/GK was 0.87 +/- 0.01 for IDR but only 0.35 +/- 0.01 for I(M) (15 mM [Rb+]o or [K+]o). The permeability sequences were generally similar for I(M) and IDR: K+ approximately Rb+ > NH4+ > Cs+, with no measurable permeability to Li+ or CH3NH3+. However, Na+ carried detectable inward current for IDR but not I(M). Nao+ also blocked inward K+ current for IDR (but not IM), at an apparent electrical distance (delta) approximately 0.4, with extrapolated dissociation constant (KD) approximately 1 M at 0 mV. Much of the instantaneous rectification of IDR in physiologic ionic conditions resulted from block by Nao+. Extracellular Cs+ carried detectable inward current for both channel types, and blocked I(M) with higher affinity (KD = 97 mM at 0 mV for I(M), KD) approximately 0.2 M at 0 mV for IDR), with delta approximately 0.9 for both. IDR showed several characteristics reflecting a multi-ion pore, including a small anomalous mole fraction effect for PRb/PK, concentration-dependent GRb/GK, and concentration- dependent apparent KD's and delta's for block by Nao+ and Cso+. I(M) showed no clear evidence of multi-ion pore behavior. For I(M), a two- barrier one-site model could describe permeation of K+ and Rb+ and block by Cso+, whereas for IDR even a three-barrier, two-site model was not fully adequate.     

Intrinsic gating of inward rectifier in bovine pulmonary artery endothelial cells in the presence or absence of internal Mg2+

Inward rectifier (IR) currents were studied in bovine pulmonary artery endothelial cells in the whole-cell configuration of the patch-clamp technique with extracellular K+ concentrations, [K+]o, ranging from 4.5 to 160 mM. Whether the concentration of free Mg2+ in the intracellular solution, [Mg2+]i, was 1.9 mM or nominally 0, the IR exhibited voltage- and time-dependent gating. The IR conductance was activated by hyperpolarization and deactivated by depolarization. Small steady-state outward IR currents were present up to approximately 40 mV more positive than the K+ reversal potential, EK, regardless of [Mg2+]i. Modeled as a first-order C in equilibrium O gating process, both the opening rate, alpha, and the closing rate, beta, were exponentially dependent on voltage, with beta more steeply voltage dependent, changing e-fold for 9 mV compared with 18 mV for an e-fold change in alpha. Over all [K+]o studied, the voltage dependence of alpha and beta shifted along with EK, as is characteristic of IR channels in other cells. The steady-state voltage dependence of the gating process was well described by a Boltzmann function. The half-activation potential was on average approximately 7 mV negative to the observed reversal potential in all [K+]o regardless of [Mg2+]i. The activation curve was somewhat steeper when Mg-free pipette solutions were used (slope factor, 4.3 mV) than when pipettes contained 1.9 mM Mg2+ (5.2 mV). The simplest interpretation of these data is that IR channels in bovine pulmonary artery endothelial cells have an intrinsic gating mechanism that is not due to Mg block.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inward rectification in rat olfactory receptor neurons.

Inwardly rectifying currents in enzymically dissociated olfactory receptor neurons of rat were studied by using patch-clamp techniques. Upon hyperpolarization to membrane potentials more negative than -100 mV, small inward-current relaxations were observed. Activation was described by a single exponential with a time constant that decreased e-fold for a 21 mV hyperpolarization. The current was not reduced by the external application of 5 mM Ba2+, but was abolished by the addition of 5 mM Cs+ to the bath solution. Increasing the external K+ concentration ([K+]o) to 25 mM dramatically enhanced the current without affecting the voltage range or the kinetics of activation. In 25 mM [K+]o, tail currents reversed at -26 mV, significantly more positive than the K+ equilibrium potential of -44 mV. These characteristics are consistent with those of a mixed Na+/K+ inward rectification that has been reported in several types of neuronal, cardiac and smooth muscle cells. The current may contribute to controlling cell excitability during the response to some odorants.     

Mytilus inhibitory peptide (MIP) induces a Na+-activated K+-current in snail neurons

Two microelectrode voltage-clamp and single-channel recordings were performed on D-cluster neurons of snail right parietal ganglion in order to study the properties of MIP-activated potassium current. It was found that the octapeptide member of the MIP-family, ASHIPRFVa elicits an outward current, which possesses all the properties characteristic for the hexapeptide(s) inward membrane response. The main component of the peptide elicited response is highly [K+]o dependent, however the response was attenuated in Na-free extracellular saline. The peptide elicited response was mimicked by raising the [Na+]i by pressure injection of Na+ into the cell. Single channel recordings indicated that MIP-induced outward K-current is Na-dependent. The probability to find a channel in open state increases with increasing intracellular Na+-concentration. Excised inside-out patches obtained from D-neurons contained I(K(Na)) channels could be activated by exposure of the cytoplasmic face of the patch membrane to 40 mM Na+, and 40 mM Li+, as well. The single channel current amplitude at -60 mV is 15 pA and the single channel conductance is 212 pS between -80 and 0 mV. It was concluded that MIP's activate a novel type of K+-current in the snail neurons. This current is the Na-activated K+-current. The single channel properties of the MIP activated channel is in concert with I(K(Na)) data obtained on different vertebrate and invertebrate preparations.     

Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology

Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from - 100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance of approximately 35 pS in symmetrical 160 mM K+. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+ (1.0) > Rb+ (0.96) > NH4+ (0.17) > Cs+ (0.07). Slope conductance ratios were: NH4+ (1.2) > K+ (1.0) > Rb+ (0.6) > Cs+ (0.10). Extracellular Cs+ or Ba2+ each induced voltage-dependent block of K(Ca) channels, with block increasing at hyperpolarizing potentials in a manner suggesting a site of block 75% across the membrane field from the outside. K(Ca) channels were blocked by tetraethylammonium (TEA) applied externally (Kd = 40 mM), but were unaffected by 10 mM TEA applied inside by pipette perfusion. K(Ca) channels were blocked by charybdotoxin (CTX) with a half-blocking dose of 3-4 nM, but were resistant to block by noxiustoxin (NTX) at 1-100 nM. Unlike K(Ca) channels in Jurkat T cells, the K(Ca) channels of normal resting or activated T cells were not blocked by apamin. We conclude that while K(Ca) and voltage-gated K+ channels in the same cells share similarities in ion permeation, Cs+ and Ba2+ block, and sensitivity to CTX, the underlying proteins differ in structural characteristics that determine channel gating and block by NTX and TEA.