Voltage-operated potassium currents in the somatic membrane of rat dorsal root ganglion neurons: ontogenetic aspects

Whole-cell transmembrane potassium currents were studied in somatic membrane of freshly isolated rat dorsal root ganglion neurons. We defined three types of potassium currents, which were separated on the basis of their different potential dependence of activation and sensitivity to external tetraethylammonium and 4-aminopyridine. The potential dependence of kinetic and steady-state properties of a fast inactivating potassium current, a slow inactivating potassium current and a non-inactivating delayed rectifier current were described by the Hodgkin-Huxley equations. A transient fast inactivating potassium current was activated at the most negative membrane potentials and was not reduced in the presence of 10 mM tetraethylammonium in the external solution. 4-Aminopyridine (2 mM) caused an 80% inhibition of this current. The activation of the fast inactivating potassium current was properly described by fitting a single exponent raised to the fourth power. The time constant of activation changed from 4 to 1 ms in the voltage range between -30 and +40 mV. The time constant of inactivation decreased from 35 to 15 ms over the same range of potentials. Parameters for the fit of a Boltzmann equation to mean values for steady-state activation were V1/2=-20mV, k=11.8mV, and for steady-state inactivation V1/2= -85 mV, k=-9.8 mV. A transient slow inactivating potassium current had an activation threshold between -40 and -30 mV. At 2 mM 4-aminopyridine, the depression of the slow potassium current was 55%. The extracellular application of 10 mM tetraethylammonium was less effective and evoked a 40% reduction. The activation of the slow inactivating potassium current was also described by a single exponential function raised to the fourth power. The time constant of activation decreased from 12 ms at a membrane potential of -10 mV to 4 ms at the potential of 60 mV. The inactivation of slow inactivating potassium current was described by two exponents. The time constant for the fast exponent ranged from 300 ms at -20 mV to 160 ms at +60 mV. The slower exponent was also potential dependent and its time constant ranged from approximately 2600 to 1600 ms over the same potentials. Parameters for the Boltzmann equation fittings to mean values were V1/2= -12.8 mV, k=13.4 mV and V1/2= -54.6 mV, k= -12 mV for steady-state activation and inactivation, respectively. A non-inactivating delayed rectifier potassium current was activated at the most positive membrane potentials. This non-inactivating current did not change in the presence of 4-aminopyridine. Extracellular tetraethylammonium (10 mM) caused a 70% reduction of this current. The activation of the non-inactivating potassium current was described by one exponent raised to the fourth power. The time constant for activation ranged from 85 ms at -5 mV to 30 ms at 45 mV. No time-dependent inactivation was observed during 15-s testing potentials in the voltage range between 10 and +60 mV. The activation behavior was characterized by V1/2=15.3 mV, k=12.5 mV. The densities of these potassium currents were studied for three groups of animals: one, five to six and 14-15 days of postnatal development. Fifty cells were examined in each age group. All three types of potassium currents were found in each investigated neuron. The mean densities of slow and fast inactivating potassium currents increased during ontogenetic development. The densities of non-inactivating delayed rectifier potassium current decreased in the first week of ontogenetic development and did not change thereafter.     

Inactivation of macroscopic late Na+ current and characteristics of unitary late Na+ currents in sensory neurons

Na+ currents in adult rat large dorsal root ganglion neurons were recorded during long duration voltage-clamp steps by patch clamping whole cells and outside-out membrane patches. Na+ current present >60 ms after the onset of a depolarizing pulse (late Na+ current) underwent partial inactivation; it behaved as the sum of three kinetically distinct components, each of which was blocked by nanomolar concentrations of tetrodotoxin. Inactivation of one component (late-1) of the whole cell current reached equilibrium during the first 60 ms; repolarizing to -40 or -50 mV from potentials of -30 mV or more positive gave rise to a characteristic increase in current (tau >/= 5 ms), attributed to removal of inactivation. A second component (late-2) underwent slower inactivation (tau > 80 ms) at potentials more positive than -80 mV, and steady-state inactivation appeared complete at -30 mV. In small membrane patches, bursts of brief openings (gamma = 13-18 pS) were usually recorded. The distribution of burst durations indicated that two populations of channel were present with inactivation rates corresponding to late-1 and late-2 macroscopic currents. The persistent Na+ current in the whole cell that extended to potentials more positive than -30 mV appeared to correspond to sporadic, brief openings that were recorded in patches (mean open time approximately 0.1 ms) over a wide potential range. None of the three types of gating described corresponded to activation/inactivation gating overlap of fast transient currents.     

The American Journal of Ophthalmology 1862-1864

A hyperpolarization-activated current (termed I[h]) is believed to provide a pacemaker depolarization in sinoatrial node cells and in some central and peripheral neurons. In the present study, we examined if such an inward cation current exists in primary auditory neurons using the whole-cell patch-clamp technique. A large inward, non-inactivating current was seen during hyperpolarizing steps negative to the resting potential. A depolarizing sag occurred during hyperpolarizing current injection, and upon termination of the current injection there was an overshoot, or a rebound firing. A low concentration of Cs+, but not Ba2+, reversibly blocked the inward current and depolarizing sag. The activation of the current showed voltage dependence with half-activation occurring at -101 +/- 1 mV. The time course of I(h) activation was fitted by double exponential function and was voltage-dependent (time constants: tau1 and tau2 = 480 and 3125 ms at -100 mV, and 66 and 404 ms at -160 mV). The reversal potential of the current was -36 mV measured from tail currents. The conductance of the current was decreased in Na+-free solution, and increased in high K+ solution. Increases in the levels of intracellular cAMP or cGMP enhanced the current. The results suggest that there exists a hyperpolarization-activated inward cation current in mammalian primary auditory neurons. This current may provide a depolarizing current during the membrane hyperpolarization following each firing of the primary auditory nerve.     

The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis

Enzymatically isolated myocytes from ferret right ventricles (12-16 wk, male) were studied using the whole cell patch clamp technique. The macroscopic properties of a transient outward K+ current I(to) were quantified. I(to) is selective for K+, with a PNa/PK of 0.082. Activation of I(to) is a voltage-dependent process, with both activation and inactivation being independent of Na+ or Ca2+ influx. Steady-state inactivation is well described by a single Boltzmann relationship (V1/2 = -13.5 mV; k = 5.6 mV). Substantial inactivation can occur during a subthreshold depolarization without any measurable macroscopic current. Both development of and recovery from inactivation are well described by single exponential processes. Ensemble averages of single I(to) channel currents recorded in cell-attached patches reproduce macroscopic I(to) and indicate that inactivation is complete at depolarized potentials. The overall inactivation/recovery time constant curve has a bell-shaped potential dependence that peaks between -10 and -20 mV, with time constants (22 degrees C) ranging from 23 ms (-90 mV) to 304 ms (-10 mV). Steady-state activation displays a sigmoidal dependence on membrane potential, with a net aggregate half-activation potential of +22.5 mV. Activation kinetics (0 to +70 mV, 22 degrees C) are rapid, with I(to) peaking in approximately 5-15 ms at +50 mV. Experiments conducted at reduced temperatures (12 degrees C) demonstrate that activation occurs with a time delay. A nonlinear least-squares analysis indicates that three closed kinetic states are necessary and sufficient to model activation. Derived time constants of activation (22 degrees C) ranged from 10 ms (+10 mV) to 2 ms (+70 mV). Within the framework of Hodgkin-Huxley formalism, Ito gating can be described using an a3i formulation.     

Isolation and characterization of a persistent potassium current in neostriatal neurons

1. Depolarization-activated, calcium-independent potassium (K+) currents were studied with the use of whole cell voltage-clamp recording from neostriatal neurons acutely isolated from adult (> or = 4 wk old) rats. The whole cell K+ current was composed of transient and persistent components. The aims of the experiments were to isolate the persistent component and then to characterize its voltage dependence and kinetics. 2. Application of 10 mM 4-aminopyridine (4-AP) completely blocked the transient currents while reducing the persistent current by approximately 40% [50% inhibitory concentration (IC50), of blockable current = 125 microM]. The persistent K+ current also was reduced by tetraethylammonium (TEA). Two components to the TEA block were present, having IC50s of 125 microM (23% of the blockable current) and 5.9 mM (77% of the blockable current). Collectively, these results suggested that the persistent components of the total K+ current was pharmacologically heterogeneous. The properties of the 4-AP-resistant, persistent K+ current (IKrp) were subsequently studied. 3. The kinetics of activation and deactivation of IKrp were voltage dependent. Examination of the entire activation/deactivation time constant profile showed that it was bell shaped, with time constants being moderately rapid (tau approximately 50 ms) at membrane potentials corresponding to the resting potential of neostriatal cells (approximately -80 mV), becoming considerably longer (tau approximately 100 ms) at potentials near the cells' spike thresholds (approximately -45 mV), and decreasing to a minimum (tau approximately 5 ms) at potentials associated with the peak of the cells' action potentials (approximately +20 mV). The inactivation kinetics of IKrp also were voltage dependent. The time constants of inactivation varied between 1 and 8 s at potentials between -10 and +35 mV. 4. Unlike persistent K+ currents in many other cell types, IKrp activated at relatively hyperpolarized membrane potentials (approximately -70 mV). The Boltzmann function describing activation had a half-activation voltage of -13 mV and a slope factor of 12 mV. In addition, the Boltzmann function describing the voltage dependence of inactivation of IKrp had a relatively depolarized half-inactivation voltage of -55 and a large slope factor of 19 mV, indicating that this current was available over a broad range of membrane potentials (between -100 and -10 mV). 5. Neostriatal neurons recorded in vivo exhibit subthreshold shifts in membrane potential of variable duration (tens of ms to s) from a hyperpolarized resting state to a depolarized state that is limited in amplitude just below spike threshold. The voltage dependence of activation and inactivation of IKrp indicates that it will be available on depolarization from the hyperpolarized state. However, the slow activation rate of this current suggests that it will contribute little either to limiting the amplitude of the initial depolarization associated with entry into the depolarized state or to depolarizing episodes of short duration (e.g., < 50 ms). However, IKrp should limit the amplitude of membrane depolarizations associated with prolonged excursions into the depolarized state.     

Calcium current and inactivation in identified neurons in Hermissenda crassicornis

1. N-type (omega-conotoxin sensitive) calcium currents (ICa) were recorded in identified neurons in Hermissenda crassicornis using low-resistance patch electrodes (0.7 +/- 0.3 M omega; n = 101) under conditions that eliminated inward Na+ currents (choline ions substitution) and suppressed outward K+ currents (Cs+, tetraethylammonium, and 4-AP). Step depolarization from a holding potential of -60 mV to potentials above -30 mV elicited ICa, which peaked approximately 20 mV and declined with increasing depolarizations. 2. Evidence for a low-threshold current was present. Step depolarization from a more hyperpolarizing potentials (e.g., -90 mV) revealed a small shoulder (< 100 pA) at -60 to -40 mV that was sensitive to Co2+ and Ni2+. However, under the conditions examined here (holding potential of -60 mV), the high-voltage-activated current predominated. 3. Barium (Ba2+) and strontium (Sr2+) permeate the Ca2+ channel with similar activation kinetics (ease of permeation; Ba2+ > Ca2+ > Sr2+). Steady-state activation of permeability versus membrane potentials for Ca2+, Ba2+, and Sr2+ as charge carriers could be fitted with the Boltzmann equation, with half-activation voltage and slope factor of 2.9 and 7.7 mV for ICa, -13.1 mV and 7.8 for Ba2+ current (IBa) and -2.3 mV and 7.8 for Sr2+ current (ISr). The time course of activation was monotonic with time constant (tau) for ICa ranging from 2 to 8 ms. 4. The inactivation profile was complex. At negative step potentials (e.g., -20 mV), inactivation of the current was slow. Depolarization steps to relatively positive voltages (e.g., 10 mV) showed more rapid inactivation than those at more positive potentials (e.g., 40 mV). When extracellular Ca2+ was raised from 5 to 10 mM, a biphasic decay (tau fast of 25 +/- 4 ms; and tau slow of 473 +/- 64 ms; mean +/- SD, n = 9) was seen. Such an observation suggested a current-mediated inactivation. 5. With a pulse duration of approximately 350 ms, ISr showed inactivation whereas Ba2+ virtually removed the decay. However, IBa turned off with more prolonged depolarization. 6. A twin-pulse protocol was used to assess the voltage dependence of inactivation: an incomplete U-shaped inactivation curve was observed for ICa, IBa, and ISr. Channels available for inactivation were increased in the presence of Ca2+ ions. 7. Inactivation was further studied with the Ca2+ chelators, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). With 10 mM of BAPTA, in the pipette, inactivation was reduced but not removed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation and inactivation of homomeric KvLQT1 potassium channels

The voltage-gated potassium channel protein KvLQT1 (Wang et al., 1996. Nature Genet. 12:17-23) is believed to underlie the delayed rectifier potassium current of cardiac muscle together with the small membrane protein minK (also named IsK) as an essential auxiliary subunit (Barhanin et al., 1996. Nature. 384:78-80; Sanguinetti et al., 1996. Nature. 384:80-83) Using the Xenopus oocyte expression system, we analyzed in detail the gating characteristics of homomeric KvLQT1 channels and of heteromeric KvLQT1/minK channels using two-electrode voltage-clamp recordings. Activation of homomeric KvLQT1 at positive voltages is accompanied by an inactivation process that is revealed by a transient increase in conductance after membrane repolarization to negative values. We studied the recovery from inactivation and the deactivation of the channels during tail repolarizations at -120 mV after conditioning pulses of variable amplitude and duration. Most measurements were made in high extracellular potassium to increase the size of inward tail currents. However, experiments in normal low-potassium solutions showed that, in contrast to classical C-type inactivation, the inactivation of KvLQT1 is independent of extracellular potassium. At +40 mV inactivation develops with a delay of 100 ms. At the same potential, the activation estimated from the amplitude of the late exponential decay of the tail currents follows a less sigmoidal time course, with a late time constant of 300 ms. Inactivation of KvLQT1 is not complete, even at the most positive voltages. The delayed, voltage-dependent onset and the incompleteness of inactivation suggest a sequential gating scheme containing at least two open states and ending with an inactivating step that is voltage independent. In coexpression experiments of KvLQT1 with minK, inactivation seems to be largely absent, although biphasic tails are also observed that could be related to similar phenomena.     

K+ channel blocking actions of flecainide compared with those of propafenone and quinidine in adult rat ventricular myocytes

The K+ channel blocking action of the class Ic antiarrhythmic agent flecainide was compared with that of propafenone and quinidine in isolated adult rat ventricular myocytes by using the whole-cell patch-clamp technique. In rat ventricular myocytes, depolarization activates both an inactivating (ITO) and a maintained (IK) outward K+ current. Flecainide, propafenone and quinidine all were potent inhibitors of ITO with IC50s of 3.7, 3.3 and 3.9 microM, respectively. Flecainide and quinidine were less potent inhibitors of IK than was propafenone with IC50s of 15 and 14 microM compared with an IC50 of 5 microM for propafenone. By contrast with their effects on outward currents, these agents produced little or no inhibition of the inward rectifier K+ current, even when present at 300 microM. All three agents produced a concentration-dependent increase in the rate of inactivation of ITO but they only produced minor hyperpolarizing shifts (approximately 3 mV) in the voltage dependence of steady-state inactivation. Although propafenone had little effect on the rate of ITO recovery from inactivation (tau CONTROL = 64 +/- 5 ms; tau PROPAFENONE = 84 +/- 9 ms), ITO recovery in the presence of flecainide and quinidine was biexponential; it exhibited an additional slow component (tau FAST = 67 +/- 5 ms and tau SLOW = 2580 +/- 1500 ms for flecainide; tau FAST = 55 +/- 5 ms and tau SLOW = 871 +/- 99 ms for quinidine). Consistent with these observations, flecainide and quinidine, but not propafenone, produced use-dependent block of ITO at a stimulation frequency of 1 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of single cardiac Na+ channels in three different gating modes

In small cell-attached patches containing one and only one Na+ channel, inactivation was studied in three different gating modes, namely, the fast-inactivating F mode and the more slowly inactivating S mode and P mode with similar inactivation kinetics. In each of these modes, ensemble-averaged currents could be fitted with a Hodgkin-Huxley-type model with a single exponential for inactivation (tauh). tauh declined from 1.0 ms at -60 mV to 0.1 ms at 0 mV in the F mode, from 4.6 ms at -40 mV to 1.1 ms at 0 mV in the S mode, and from 4.5 ms at -40 mV to 0.8 ms at +20 mV in the P mode, respectively. The probability of non-empty traces (net), the mean number of openings per non-empty trace (op/tr), and the mean open probability per trace (popen) were evaluated at 4-ms test pulses. net inclined from 30% at -60 mV to 63% at 0 mV in the F mode, from 4% at -90 mV to 90% at 0 mV in the S mode, and from 2% at -60 mV to 79% at +20 mV in the P mode. op/tr declined from 1.4 at -60 mV to 1.1 at 0 mV in the F mode, from 4.0 at -60 mV to 1.2 at 0 mV in the S mode, and from 2.9 at -40 mV to 1.6 at +20 mV in the P mode. popen was bell-shaped with a maximum of 5% at -30 mV in the F mode, 48% at -50 mV in the S mode, and 16% at 0 mV in the P mode. It is concluded that 1) a switch between F and S modes may reflect a functional change of inactivation, 2) a switch between S and P modes may reflect a functional change of activation, 3) tauh is mainly determined by the latency until the first channel opening in the F mode and by the number of reopenings in the S and P modes, 4) at least in the S and P modes, inactivation is independent of pore opening, and 5) in the S mode, mainly open channels inactivate, and in the P mode, mainly closed channels inactivate.     

Whole cell recording of sodium, potassium and calcium currents of cultured neonatal rat sympathetic neurons

Na, K and Ca currents and other electrophysiological characteristics of cultured neonatal rat superior cervical sympathetic neurons were studied using whole cell clamp technique. The mean passive and active membrane properties measured are as follows: resting membrane potential, -51 +/- 6 mV; input resistance, 1432 +/- 389 M omega; time constant, 130 +/- 32 ms; amplitude of action potential, 96 +/- 10 mV; overshoot, 42 +/- 6 mV. Na, K and Ca currents were isolated upon pharmacological manipulations. The predominant type of K current was a noninactivating delayed rectifier. Voltage-clamp studies also showed the presence of a high voltage-activated sustained inward Ca current, while low voltage could not elicit any transient Ca current.     

Action-potential-like depolarizations relieve opioid inhibition of N-type Ca2+ channels in NG108-15 cells

The ability of action-potential-like waveforms (APWs) to attenuate opioid-induced inhibition of N-type Ca2+ channels was investigated in the neuroblastoma x glioma cell line NG108-15 using whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells, the opioid agonist [d-ala2]-methionine-enkephalin (DAME) reversibly decreased omega-conotoxin-GVIA-sensitive Ba2+ currents (N-type currents). Agonist-mediated inhibition of N-type currents could be transiently relieved by strong unphysiological depolarizing prepulses to +80 mV (facilitation). Significant facilitation was also achieved by conditioning the cell with a train of 15 APWs, which roughly mimicked physiological action potentials (1- to 6-ms-long depolarizations to +30 mV from a holding potential of -40 mV). The APW-induced facilitation depended on both conditioning pulse frequency and duration. Summation of the disinhibition produced by each APW was possible because reinhibition following repolarization to -40 mV was a much slower process (tau=88 ms) than the onset of facilitation at +80 mV (tau=7 ms). These results provide evidence that N-type Ca2+ channel facilitation may be a physiologically relevant process, and suggest that neuronal firing may relieve agonist-induced inhibition of N-type currents to an extent depending on both the shape of action potentials and the frequency of firing.     

The voltage-dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of gamma-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal-Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about -45 mV, was half-maximal steady-state inactivated at -66.6 mV, and recovery from inactivation followed a two-exponential process (tau1 = 8.4 ms and tau2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated approximately -40 mV, declined rapidly with a voltage-insensitive time constant (tau approximately 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mM). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 microM) and a second by high concentrations (6 mM). After elimination of Ifast by a conditioning prepulse (50 ms to -50 mV), a slow K+ current (I(KV)) could be studied in isolation. I(KV) was only moderately affected by 4-AP (6 mM), while TEA (20 mM) blocked most (> 80%) of the current. I(KV) activated at about -40 mV, declined monoexponentially in a voltage-dependent manner (tau approximately 850 ms at -30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and I(KV), indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and I(KV) were blocked by tetrodotoxin (TTX, 1 microM), 4-AP (6 mM) and TEA (20 mM) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about -30 mV, were completely blocked by 50 microM cadmium (Cd2+), were sensitive to verapamil (approximately 40% block by 10 microM), and were not affected by nickel (50 microM). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (approximately 2 ms, measured at half maximal amplitude) of relative small amplitude (approximately 83 mV, measured from the action potential threshold).     

Potassium currents in freshly dissociated uterine myocytes from nonpregnant and late-pregnant rats

In freshly dissociated uterine myocytes, the outward current is carried by K+ through channels highly selective for K+. Typically, nonpregnant myocytes have rather noisy K+ currents; half of them also have a fast-inactivating transient outward current (ITO). In contrast, the current records are not noisy in late pregnant myocytes, and ITO densities are low. The whole-cell IK of nonpregnant myocytes respond strongly to changes in [Ca2+]o or changes in [Ca2+]i caused by photolysis of caged Ca2+ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca2+ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at -80, -40, or 0 mV and digital subtractions, the whole-cell IK of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately -61 and -22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca2+-activated K+ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca2+-sensitive K+ channels also have a half-open probability at 68 mV, and are less sensitive to Ca2+ than similar channels in taenia coli myocytes. Ca2+-activated K+ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30-35% of the total IK in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute approximately 20% of total IK in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K+ currents that include a suppression of the ITO, a redistribution of IK expression from large-conductance Ca2+-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca2+ sensitivity, and a positive shift of the activation of some large-conductance Ca2+-activated channels.     

Holistic nursing--definition and realization

Single-channel properties of a delayed rectifier voltage-gated K+ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between -60 and -40 mV with a potential of half-maximal activation, E50, at -47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between -60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at -40 mV. The time constant of deactivation was 126 msec at -80 mV and 16.9 msec at -110 mV. In symmetrical 105 mM K+, the single-channel conductance (gamma) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13-15 degrees C. In Na+ -rich solution with 2.5 mM extracellular K+ gamma was 7 pS and the reversal potential was negative to -80 mV, indicating a high selectivity for K+ over Na+. gamma depended on extracellular K+ concentration (KD = 19.6 mM) and temperature (Q10 = 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel amplitude at all potentials tested with a half-maximal inhibiting concentration (IC50) of 0.6 mM. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC50 = 6.8 nM). mast cell degranulating peptide (MCDP, IC50 = 41.9 microM). In Ringer solution the membrane potential of macroscopic I-channel patches was about -65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential.     

Rates and equilibria at the acetylcholine receptor of Electrophorus electroplaques: a study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, alpha, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/tau as the sum of the rate constant alpha for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate alpha, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to alpha-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).     

Gating of I(sK) channels expressed in Xenopus oocytes

The channel underlying the slow component of the voltage-dependent delayed outward rectifier K+ current, I(Ks), in heart is composed of the minK and KvLQT1 proteins. Expression of the minK protein in Xenopus oocytes results in I(Ks)-like currents, I(sK), due to coassembly with the endogenous XKvLQT1. The kinetics and voltage-dependent characteristics of I(sK) suggest a distinct mechanism for voltage-dependent gating. Currents recorded at 40 mV from holding potentials between -60 and -120 mV showed an unusual "cross-over," with the currents obtained from more depolarized holding potentials activating more slowly and deviating from the Cole-Moore prediction. Analysis of the current traces revealed two components with fast and slow kinetics that were not affected by the holding potential. Rather, the relative contribution of the fast component decreased with depolarized holding potentials. Deactivation and reactivation, after a short period of repolarization (100 ms), was markedly faster than the fast component of activation. These gating properties suggest a physiological mechanism by which cardiac I(Ks) may suppress premature action potentials.     

When management works: an organizational culture that facilitates learning to self-manage type 2 diabetes

Endogenous voltage-gated potassium currents were investigated in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells using whole-cell voltage clamp recording. Depolarizing voltage steps from -70 mV triggered an outwardly rectified current in nontransfected HEK293 cells. This current had an amplitude of 296 pA at +40 mV and a current density of 19.2 pA/pF. The outward current was eliminated by replacing internal K+ with Cs+ and suppressed by the K+ channel blockers tetraethylammonium and 4-aminopyridine. Raising external K+ attenuated the outward current and shifted the reversal potential towards positive potentials as predicted by the Nernst equation. The current had a fast activation phase but inactivated slowly. These features implicate delayed rectifier (I(K))-like channels as mediators of the observed current, which was comparable in size to I(K) currents in many other cells. A small native inward rectifier current but no transient outward current I(A), the M current I(M), or Ca2+-dependent K+ currents were detected in HEK293 cells. In contrast to these findings in HEK293 cells, little or no I(K)-like current was detected in CHO cells. The difference in endogenous voltage-activated currents in HEK293 and CHO cells suggest that CHO cell lines are a preferred system for exogenous K+ channel expression.     

Putative M-type potassium channels in neuroblastoma-glioma hybrid cells: inhibition by muscarine and bradykinin

Putative M-type K(+)-channels ('M-channels') were recorded in differentiated NG108-15 neuroblastoma x glioma hybrid cells transformed to express m1 muscarinic acetylcholine receptors using cell-attached patch-electrodes. Channels showed multiple conductances, with peaks at 6-9 and 12-15 pS. Averaged currents showed time-dependent activation during 1 s depolarization steps to around -30 mV. Steady-state Po increased in a voltage-dependent manner when the membrane was depolarized between 10 and 60 mV, with a limiting slope of 5.5 mV/e-fold change in Po. Steady-state kinetics were fit by two open and three shut times: depolarization shortened shut times and lengthened open times. Application of muscarine (10 microM) or bradykinin (10 microM) to the membrane outside the patch reversibly reduced steady-state in-patch channel activity to 38.4 +/- 11.7 and 28.8 +/- 6.1% of control values, respectively. Inhibition was accompanied by a lengthening of channel shut times without significant change in open times or distribution of conductance levels. No effect of muscarine or bradykinin on whole-cell or membrane patch delayed rectifier currents was detected. It is concluded that M-channels in NG108-15 cells are qualitatively similar to, but sparser than, those previously reported in rat sympathetic neurones. Their inhibition by extra-patch acetylcholine and bradykinin suggests that a mobile messenger is involved in the transduction process leading from receptor activation to channel closure.     

Time-dependent outward currents through the inward rectifier potassium channel IRK1. The role of weak blocking molecules

Outward currents through the inward rectifier K+ channel contribute to repolarization of the cardiac action potential. The properties of the IRK1 channel expressed in murine fibroblast (L) cells closely resemble those of the native cardiac inward rectifier. In this study, we added Mg2+ (0.44-1.1 mM) or putrescine (approximately 0.4 mM) to the intracellular milieu where endogenous polyamines remained, and then examined outward IRK1 currents using the whole-cell patch-clamp method at 5.4 mM external K+. Without internal Mg2+, small outward currents flowed only at potentials between -80 (the reversal potential) and approximately -40 mV during voltage steps applied from -110 mV. The strong inward rectification was mainly caused by the closed state of the activation gating, which was recently reinterpreted as the endogenous-spermine blocked state. With internal Mg2+, small outward currents flowed over a wider range of potentials during the voltage steps. The outward currents at potentials between -40 and 0 mV were concurrent with the contribution of Mg2+ to blocking channels at these potentials, judging from instantaneous inward currents in the following hyperpolarization. Furthermore, when the membrane was repolarized to -50 mV after short depolarizing steps (> 0 mV), a transient increase appeared in outward currents at -50 mV. Since the peak amplitude depended on the fraction of Mg(2+)-blocked channels in the preceding depolarization, the transient increase was attributed to the relief of Mg2+ block, followed by a re-block of channels by spermine. Shift in the holding potential (-110 to -80 mV), or prolongation of depolarization, increased the number of spermine-blocked channels and decreased that of Mg(2+)-blocked channels in depolarization, which in turn decreased outward currents in the subsequent repolarization. Putrescine caused the same effects as Mg2+. When both spermine (1 microM, an estimated free spermine level during whole-cell recordings) and putrescine (300 microM) were applied to the inside-out patch membrane, the findings in whole-cell IRK1 were reproduced. Our study indicates that blockage of IRK1 by molecules with distinct affinities, spermine and Mg2+ (putrescine), elicits a transient increase in the outward IRK1, which may contribute to repolarization of the cardiac action potential.     

Gating of mammalian cardiac gap junction channels by transjunctional voltage

Numerous two-cell voltage-clamp studies have concluded that the electrical conductance of mammalian cardiac gap junctions is not modulated by the transjunctional voltage (Vj) profile, although gap junction channels between low conductance pairs of neonatal rat ventricular myocytes are reported to exhibit Vj-dependent behavior. In this study, the dependence of macroscopic gap junctional conductance (gj) on transjunctional voltage was quantitatively examined in paired 3-d neonatal hamster ventricular myocytes using the double whole-cell patch-clamp technique. Immunolocalization with a site-specific antiserum directed against amino acids 252-271 of rat connexin43, a 43-kD gap junction protein as predicted from its cDNA sequence, specifically stained zones of contact between cultured myocytes. Instantaneous current-voltage (Ij-Vj) relationships of neonatal hamster myocyte pairs were linear over the entire voltage range examined (0 less than or equal to Vj less than or equal to +/- 100 mV). However, the steady-state Ij-Vj relationship was nonlinear for Vj greater than +/- 50 mV. Both inactivation and recovery processes followed single exponential time courses (tau inactivation = 100-1,000 ms, tau recovery approximately equal to 300 ms). However, Ij recovered rapidly upon polarity reversal. The normalized steady-state junctional conductance-voltage relationship (Gss-Vj) was a bell-shaped curve that could be adequately described by a two-state Boltzmann equation with a minimum Gj of 0.32-0.34, a half-inactivation voltage of -69 and +61 mV and an effective valence of 2.4-2.8. Recordings of gap junction channel currents (ij) yielded linear ij-Vj relationships with slope conductances of approximately 20-30 and 45-50 pS. A kinetic model, based on the Boltzmann relationship and the polarity reversal data, suggests that the opening (alpha) and closing (beta) rate constants have nearly identical voltage sensitivities with a Vo of +/- 62 mV. The data presented in this study are not consistent with the contingent gating scheme (for two identical gates in series) proposed for other more Vj-dependent gap junctions and alternatively suggest that each gate responds to the applied Vj independently of the state (open or closed) of the other gate.