Internal TEA Research Articles

Gating currents in Shaker K+ channels. Implications for activation and inactivation models

We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel.

Probing of pore in the Chara gymnophylla K + channel by blocking cations and by streaming potential measurements

The current—voltage (I–V) relationship of single K + channels present in the Chara gymnophylla droplet membrane was studied. The channel presumably contains large mouths at both pore ends which are sufficiently wide to accomodate TEA + as evidenced by internal and external TEA + blockade. The voltage dependence of blockade by external Cs + and Na + reveals the multi-ion occupancy of the channel. The value of streaming potential (4.0 mV/osmol) measured in the Chara K + channel indicates that the channel contains up to nine water molecules in the narrow region. It is concluded that the length of this region is around 28 Å.

On the interaction of bovine pancreatic trypsin inhibitor with maxi Ca(2+)-activated K+ channels. A model system for analysis of peptide-induced subconductance states.

Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue basic peptide that is a representative member of a widely distributed class of serine protease inhibitors known as Kunitz inhibitors. BPTI is also homologous to dendrotoxin peptides from mamba snake venom that have been characterized as inhibitors of various types of voltage-dependent K+ channels. In this study we compared the effect of DTX-I, a dendrotoxin peptide, and BPTI on large conductance Ca(2+)-activated K+ channels from rat skeletal muscle using planar bilayer methodology. As previously found for DTX-I (1990. Neuron. 2:141-148), BPTI induces the appearance of distinct subconductance events when present on the internal side of maxi K(Ca) channels. The single channel kinetics of substate formation follow the predictions of reversible binding of the peptide to a single site or class of sites with a Kd of 4.6 microM at 0 mV and 50 mM symmetrical KCl. The apparent association rate of BPTI binding decreases approximately 1,000-fold per 10-fold increase in ionic strength, suggestive of a strong electrostatic interaction between the basic peptide and negative surface charge in the vicinity of the binding site. The equilibrium Kd for BPTI and DTX-I is also voltage dependent, decreasing e-fold per 30 mV of depolarization. The unitary subconductance current produced by BPTI binding exhibits strong inward rectification in the presence of symmetrical KCl, corresponding to 15% of open channel current at +60 mV and 70% of open state at -40 mV. In competition experiments, the internal pore-blocking ions, Ba2+ and TEA+, readily block the substate with the same affinity as that for blocking the normal open state. These results suggest that BPTI does not bind near the inner mouth of the channel so as to directly interfere with cation entry to the channel. Rather, the mechanism of substate production appears to involve a conformational change that affects the energetics of K+ permeation.

Electrophysiological Study on a Cochlear Outer Hair Cell

In order to examine the mechanoelectrical transduction of a cochlear hair cell, the electrical characteristics of the cochlear outer hair cell were studied. Both the cell dissociation technique and the patch clamp technique were used in this experiment. The membrane current gained by the voltage clamp method had a biphasic component, an initial inward current and a following outward current. After blocking the outward current by internal Cs ion and external TEA, the inward current has voltage-dependent inactivation. Also, the membrane potential change was studied by the current clamp method.

Multiple blocking mechanisms of ATP-sensitive potassium channels of frog skeletal muscle by tetraethylammonium ions.

1. Patch-clamp methods were used to study the action of tetraethylammonium ions (TEA+) and other quaternary ammonium ions on adenosine-5'-triphosphate (ATP)-sensitive K+ channels in sarcolemmal vesicles from frog skeletal muscle. The blocking ions were applied either to the external or the internal surface of the membrane patch. 2. External TEA+ caused a very fast block, so that the amplitude of single-channel currents was reduced. Open-channel variance was decreased. The block was 1:1, with a dissociation constant (Kd) of 6-7 mM. We could detect no voltage dependence of Kd. 3. External TEA+ prolonged open times in a manner consistent with the channel being unable to close when blocked by TEA+. 4. TEA+ also blocked when applied to the internal side of the membrane. This block showed two components with different kinetics and different affinities. The slow block chopped up openings into much briefer events and had a Kd of about 1.4 mM at -3 mV. The fast block reduced the amplitude of unitary currents and was of lower affinity, with Kd around 26 mM. 5. The slow block by internal TEA+ was markedly voltage dependent, the Kd decreasing e-fold for a 37 mV depolarization. Both the association and dissociation rates were dependent on voltage. In contrast, the fast block by internal TEA+ appeared virtually independent of voltage. 6. The effects of internally applied tetramethylammonium (TMA+) and tetrapentylammonium (TPA+) ions were also investigated. Internal TMA+ produced a flickery block while the block by internal TPA+ was similar to that caused by TEA+, although TPA+ was about 10 times more effective. 7. Our results suggest that the channel has three binding sites for TEA+, one of which is accessible from the outside of the membrane. Only one of the internal sites is located so as to experience a substantial fraction of the membrane voltage field.

Asymmetric charge movement and calcium currents in ventricular myocytes of neonatal rat.

1. Calcium and sodium currents and non-linear capacitive currents were recorded from isolated ventricular cells from neonatal rats, using the whole-cell patch-clamp technique, usually with a holding potential of -100 mV. 2. When recording with internal and external solutions designed to suppress virtually all ionic currents except the calcium current, careful subtraction of all linear capacitive and ionic currents revealed that depolarizations elicited a small transient outward current which preceded the inward calcium current. This outward current was discernible just below the threshold potential for the calcium current and increased with larger depolarizations to a maximum for potentials of about +30 mV and above. 3. Elimination of the calcium current revealed that at each potential the transient outward current was accompanied by a roughly equal transient inward current upon repolarization. The properties of these currents indicate that they are non-linear capacitive currents. Best-fit Boltzmann curves of the 'on' charge (integral of the transient outward current) gave values for qmax, V and k of 3.9 nC/microF, -29.3 mV and 15.5 mV with internal Cs+. The maximum 'on' charge is similar to that found with calcium currents (4.3 nC/microF). Similar values were obtained with internal TEA+. 4. Boltzmann fits of conductance vs. voltage for the calcium channel gave mean values of -15.5 and 13.3 mV for V and k (with internal Cs+); the corresponding values for the sodium channel were -49.9 and 5.4 mV. 5. Pre-pulses (20 ms) to -60 mV inactivated 77% of the peak sodium current, but only inactivated about 10% of the peak calcium current and reduced the maximum 'on' charge (moved at potentials positive to -60 mV) by 19%. 6. With a holding potential of -100 mV, 10 microM-nifedipine blocked 89% of the calcium current, but had little effect on the amount of 'on' charge. The 'off' charge appeared to be slower in the presence of nifedipine. 7. These results and consideration of the number of calcium channels and high-affinity binding sites for dihydropyridines (DHP), suggest that a large part of the charge movement may be related to DHP binding sites and involved with gating calcium channels. Comparison with skeletal muscle suggests similarities in the mechanisms involved in excitation-contraction coupling.

Na currents and action potentials in rat myelinated nerve fibres at 20 and 37° C

(1) Action potentials and membrane currents were recorded in single myelinated rat nerve fibres at 20 and 37 degrees C. Three experiments were also performed in single cat nerve fibres. (2) K currents were blocked by internal CsCl and external TEA. The steady state and kinetic parameters of Na activation and inactivation were determined at both temperatures. (3) When the temperature was raised from 20 to 37 degrees C, steady state Na activation, m infinity (V), and inactivation, h infinity (V), did not change significantly. (4) The time constant of Na activation, tau m, was determined within the potential range of -40 to 125 mV at 20 degrees C and V = 40-60 mV at 37 degrees C. The temperature coefficient, Q10, of tau m was 2.2. (5) The decay in the Na current was described by two exponentials at both temperatures. The amplitude of the slow phase was 1-10%. The time constant of the fast phase of Na inactivation, tau h1, was determined at both temperatures within the potential range of -50 mV to 125 mV. The Q10 of tau h1 was 2.9 and did not depend on potential. (6) The Na equilibrium potential was 152 mV at 20 degrees C and 144 mV at 37 degrees C. The leakage conductance was 24 nS at 20 degrees C and 43 nS at 37 degrees C. These differences were interpreted as signs of fibre deterioration at higher temperature. (7) The results from the current and voltage clamp experiments performed in the cat nerve were essentially the same as those in the rat nerve fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium currents in internally perfused nerve cell bodies of Limnea stagnalis.

1. When K(+) is removed from both sides of the somal membrane of Limnea neurones, time-dependent, voltage-dependent outward currents are observed at positive potentials. These currents can be carried by Tris(+) and tetraethylammonium (TEA(+)), as well as Cs(+), but the Cs currents are several times larger. The Cs currents are not affected by external or internal TEA, but are strongly reduced by 4-aminopyridine (4-AP) and all Ca blockers tried.2. The presence of these non-specific outward currents and their sensitivity to all treatments that eliminate the Ca currents prevent the complete isolation of Ca currents. The non-specific outward currents are most prominent at large positive potentials and as slow tail currents on stepping back to the holding potential.3. Ca currents are ;washed out' in well perfused cells. Typically the Ca current has decayed to less than one tenth of its original size after (1/2) h of perfusion. This wash-out is specific for the Ca current; Na and K currents persist for several hours.4. Once the Ca current has completely decayed, it is possible to study one type of non-specific current without overlapping inward currents. This current activates between 0 and +30 mV and appears to reverse near 0 mV.5. In spite of the probable presence of slowly activating outward currents, the net inward currents measured show little apparent inactivation. In all the cells studied the inward current evoked at +20 mV has never decayed by more than 50% during a 60 ms pulse. So the true inactivation of these Ca currents must be quite slow, with time constants of the order of 100 ms and larger.6. The activation of the Ca current agrees with m(2) kinetics. The rate of activation is the same for Ba currents as for Ca currents.7. When the membrane potential is stepped back to the holding level (-50 mV), the Ca current turns off quite rapidly with a time constant of about 100 mus (25 degrees C). The time constant for turning off the Ca current is not related to the time constant for turning on the Ca current at the same voltage as expected for m(2) kinetics in the Hodgkin and Huxley model. At -30 mV the tau(m) for turn-on is eight times larger than the tau(m) for turn-off.

Evidence for the presence of potassium channels in the paranodal region of acutely demyelinated mammalian single nerve fibres.

1. A study has been made of the ionic currents in voltage-clamped single rabbit nodes of Ranvier at 22-26 degrees C both under normal conditions, and after the nerve fibres had been acutely demyelinated by a variety of treatments designed to lossen the myelin from the axonal membrane. 2. The myelin-loosening treatments included application of various combinations of: lysolecithin (to dissolve the myelin); collagenase (to loosen the connective tissue in the nodal region); high-potassium Locke solution, hypertonic and hypotonic solutions (to induce axonal volume changes). 3. At a critical stage in such treatment (usually after 15-45 min) a large outward current suddenly appeared. 4. There was no substantial change in the size of the measured inward sodium current when measured at this critical stage. 5. The outward current was blocked by internal TEA and caesium ions, had a reversal potential that became more positive when the external potassium concentration was increased, was kinetically similar to the known potassium current in frog fibres, and was therefore assumed to be a potassium current. 6. The phase of large outward current, whenever it appeared, was always accompanied by the appearance of a slow transient capacitative component in the leakage current, which indicated a marked increase in the effective nodal capacity (of 10- to 60-fold). We suggest that the slow transient capacity current reflected charging of newly exposed axonal membrane, probably in the paranodal region, which was uncovered by the various acute demyelination treatments. This internodal membrane seems to contain mostly potassium channels and few, if any, sodium channels. 7. Newly dissected fibres occasionally showed large potassium currents before treatment, particularly if they were deliberately stretched during dissection; a marked slow capacity transient current was consistently present in these fibres. 8. The effects of acute paranodal demyelination on the sodium and potassium currents, and on the transient capacity currents, can be simulated by a model in which the node is coupled to a cable-like paranode which contains Hodgkin--Huxley type potassium channels and which has a much higher leakage resistance. 9. The functional significance of the presence of potassium channels in rhe internodal region (at least in the paranode) of mammalian fibres is discussed.

Chemically induced K+ conduction noise in squid axon.

Internal perfusion of tetraethylammonium ions (TEA) in squid axons produces a significant high frequency noise component. Although internal TEA suppresses the potassium conductance (GK) noise at relatively low frequencies, it induces high frequency noise which exceeds the intensity of the normal potassium and sodium noise. In addition, the induced noise is dependent on the presence of internal potassium ions (K+) suggesting that this source of noise arises from a modulation of the K+ conductance due to the blocking and unblocking of the K+ channel. The simplest model describing the TEA data is a two-step sequential, pseudo-unimolecular reaction where TEA binds during an open conductance state. A unit channel conductance of 2 pS is estimated from the TEA data as well as noise induced by triethyldecylammonium (TEDA) ions. Thus, these data are consistent with the hypothesis that the channel is blocked whenever the quaternary ammonium ion binding site, located near or within the K+ channel, is occupied.