Voltage-gated K+ channels exhibit a slow inactivation process, which becomes an important influence on the rate of action potential repolarization during prolonged or repetitive depolarization. During slow inactivation, the outer mouth of the permeation pathway undergoes a conformational change. We report here that during the slow inactivation process, the channel progresses through at least three permeation states; from the initial open state that is highly selective for K+, the channel enters a state that is less permeable to K+ and more permeable to Na+, and then proceeds to a state that is non-conducting. Similar results were obtained in three different voltage-gated K+ channels: Kv2.1, a channel derived from Shaker (Shaker Delta A463C), and a chimeric channel derived from Kv2.1 and Kv1.3 that displays classical C-type inactivation. The change in selectivity displayed both voltage- and time-dependent properties of slow inactivation and was observed with K+ on either side of the channel. Elevation of internal [K+] inhibited Na+ conduction through the inactivating channel in a concentration-dependent manner. These results indicate that the change in selectivity filter function is an integral part of the slow inactivation mechanism, and argue against the hypothesis that the inactivation gate is independent from the selectivity filter. Thus, these data suggest that the selectivity filter is itself the inactivation gate.

The voltage-gated K+ channel, Kv2.1, conducts Na+ in the absence of K+. External tetraethylammonium (TEAo) blocks K+ currents through Kv2.1 with an IC50 of 5 mM, but is completely without effect in the absence of K+. TEAo block can be titrated back upon addition of low [K+]. This suggested that the Kv2.1 pore undergoes a cation-dependent conformational rearrangement in the external vestibule. Individual mutation of lysine (Lys) 356 and 382 in the outer vestibule, to a glycine and a valine, respectively, increased TEAo potency for block of K+ currents by a half log unit. Mutation of Lys 356, which is located at the outer edge of the external vestibule, significantly restored TEAo block in the absence of K+ (IC50 = 21 mM). In contrast, mutation of Lys 382, which is located in the outer vestibule near the TEA binding site, resulted in very weak (extrapolated IC50 = ∼265 mM) TEAo block in the absence of K+. These data suggest that the cation-dependent alteration in pore conformation that resulted in loss of TEA potency extended to the outer edge of the external vestibule, and primarily involved a repositioning of Lys 356 or a nearby amino acid in the conduction pathway. Block by internal TEA also completely disappeared in the absence of K+, and could be titrated back with low [K+]. Both internal and external TEA potencies were increased by the same low [K+] (30–100 μM) that blocked Na+ currents through the channel. In addition, experiments that combined block by internal and external TEA indicated that the site of K+ action was between the internal and external TEA binding sites. These data indicate that a K+-dependent conformational change also occurs internal to the selectivity filter, and that both internal and external conformational rearrangements resulted from differences in K+ occupancy of the selectivity filter. Kv2.1 inactivation rate was K+ dependent and correlated with TEAo potency; as [K+] was raised, TEAo became more potent and inactivation became faster. Both TEAo potency and inactivation rate saturated at the same [K+]. These results suggest that the rate of slow inactivation in Kv2.1 was influenced by the conformational rearrangements, either internal to the selectivity filter or near the outer edge of the external vestibule, that were associated with differences in TEA potency.

Intracellular and extracellular recordings were made from pyramidal neurons in hippocampal slices in order to study spontaneous paroxysmal bursting induced by raising the extracellular potassium concentration from 3.5 to 8.5 mM. Extracellular recordings from all hippocampal subfields indicated that spontaneous bursts appeared to originate in region CA3c or CA3b as judged by burst onset. Burst intensity was also greatest in regions CA3b and CA3c and became progressively less toward region CA2. Intracellular recordings indicated that in 8.5 mM potassium, large spontaneous excitatory postsynaptic potentials (EPSPs), large burst afterhyperpolarizations, and rhythmic hyperpolarizing-depolarizing waves of membrane potential were invariably present in CA3c neurons. High potassium (8.5 mM) induced a positive shift (+9 mV) in the reversal potential of GABAergic inhibitory postsynaptic potentials (IPSPs) in CA3c neurons without changing input resistance or resting potential. This resulted in a drastic reduction in amplitude of the IPSP. Reduction of IPSP amplitude occurred before the onset of spontaneous bursting and was reversible upon return to normal potassium. A new technique to quantify the relative intensity of interictal-like burst discharges is described. Pentobarbital, diazepam, and GABA uptake inhibitors, which enhance GABA-mediated synaptic inhibition, reduced the intensity of potassium-induced bursts, whereas the GABA antagonist bicuculline increased burst intensity. Diphenylhydantoin and phenobarbital, anticonvulsants that have little effect on GABAergic inhibition, were without effect on spontaneous bursts. Burst frequency was reduced by bicuculline and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol but was unaffected by other drugs. Reduction of slice temperature from 35 to 19 degrees C dramatically reduced burst intensity but did not markedly affect burst frequency. We hypothesize that high potassium induces a rise in intracellular chloride concentration, possibly by activating an inward KCl pump or by a passive Donnan effect, which results in a decreased IPSP amplitude. With inhibition suppressed, the large spontaneous EPSPs that appear in high potassium cause individual CA3c neurons to fire. A combination of synaptic and electrical interactions among CA3c cells then synchronizes discharges into interictal spike bursts.

With prolonged or repetitive activation, voltage-gated K+ channels undergo a slow (C-type) inactivation mechanism, which decreases current flow through the channel. Previous observations suggest that C-type inactivation results from a localized constriction in the outer mouth of the channel pore and that the rate of inactivation is controlled by the-rate at which K+ leaves an unidentified binding site in the pore. We have functionally identified two K+ binding sites in the conduction pathway of a chimeric K+ channel that conducts Na+ in the absence of K+. One site has a high affinity for K+ and contributes to the selectivity filter mechanism for K+ over Na+. Another site, external to the high-affinity site, has a lower affinity for K+ and is not involved in channel selectivity. Binding of K+ to the high-affinity binding site slowed inactivation. Binding of cations to the external low-affinity site did not slow inactivation directly but could slow it indirectly, apparently by trapping K+ at the high-affinity site. These data support a model whereby C-type inactivation involves a constriction at the selectivity filter, and the constriction cannot proceed when the selectivity filter is occupied by K+.

Permeation selectivity was studied in two human potassium channels, Kv2.1 and Kv1.5, expressed in a mouse cell line. With normal concentrations of potassium and sodium, both channels were highly selective for potassium. On removal of potassium, Kv2.1 displayed a large sodium conductance that was inhibited by low concentrations of potassium. The channel showed a competition mechanism of selectivity similar to that of calcium channels. In contrast, Kv1.5 displayed a negligible sodium conductance on removal of potassium. The observation that structurally similar potassium channels show different abilities to conduct sodium provides a basis for understanding the structural determinants of potassium channel selectivity.

SUMMARY1. Intracellular recordings were made from CAI pyramidal cells in the rat hippocampal slice to study the processes that influence the time course of inhibitory post-synaptic potentials (i.p.s.p.s) mediated by y-aminobutyric acid (GABA), and conductance changes evoked by ionophoretically applied GABA.2. The GABA-uptake inhibitors, nipecotic acid and cis-4-OH-nipecotic acid (1 mM), greatly prolonged conductance increases associated with both hyperpolarizing and depolarizing responses to ionophoretically applied GABA. In contrast to their effects on GABA-evoked conductances, uptake inhibitors only slightly

The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca ++ exchange on rundown of voltage-gated Ca ++ currents and on the duration of tail currents mediated by Ca § CI-channels. Ca §247 currents were studied in GH3 pituitary cells; Ca++-dependent CI-currents were studied in AtT-20 pituitary cells. Na+-Ca ++ exchange was inhibited by substitution of tetraethylammonium (TEA +) or tetramethylammonium (TMA +) for extracellular Na § Control experiments demonstrated that substitution of TEA § for Na § did not produce its effects via a direct interaction with Ca+ +-dependent CI-channels or via blockade of Na+-H + exchange. When studied with standard whole cell methods, Ca § § and Ca + +-dependent C1-currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca + § exchange. In contrast, the amplitude of both Ca + § and Ca § dependent CI-currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na § § exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca+ § dent CI-current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca + § buffering mechanisms remained stable. The duration of Ca §247 CI-currents was positively correlated with the amount of Ca + § influx through voltage-gated Ca § channels, and was prolonged by inhibition of Na § §247 exchange. The influence of Na+-Ca ++ exchange on CI-currents was greater for larger currents, which were produced by greater influx of Ca §247 . Regardless of Ca §247 influx, however, the prolongation of Cl-tail currents that resulted from inhibition of Na+-Ca + § exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na § to TEA+-containing bath solutions.

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