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Voltage-gated potassium channels such as Shaker help to control electrical signalling in neurons by regulating the passage of K+ across cell membranes. Ion flow is controlled by a voltage-dependent gate at the intracellular side of the pore, formed by the crossing of four alpha-helices--the inner-pore helices. The prevailing model of gating is based on a comparison of the crystal structures of two bacterial channels--KcsA in a closed state and MthK in an open state--and proposes a hinge motion at a conserved glycine that splays the inner-pore helices wide open. We show here that two types of intersubunit metal bridge, involving cysteines placed near the bundle crossing, can occur simultaneously in the open state. These bridges provide constraints on the open Shaker channel structure, and on the degree of movement upon opening. We conclude that, unlike predictions from the structure of MthK, the inner-pore helices of Shaker probably maintain the KcsA-like bundle-crossing motif in the open state, with a bend in this region at the conserved proline motif (Pro-X-Pro) not found in the bacterial channels. A narrower opening of the bundle crossing in Shaker K+ channels may help to explain why Shaker has an approximately tenfold lower conductance than its bacterial relatives.

The structure of the bacterial potassium channel KcsA has provided a framework for understanding the related voltage-gated potassium channels (Kv channels) that are used for signalling in neurons. Opening and closing of these Kv channels (gating) occurs at the intracellular entrance to the pore, and this is also the site at which many open channel blockers affect Kv channels. To learn more about the sites of blocker binding and about the structure of the open Kv channel, we investigated here the ability of blockers to protect against chemical modification of cysteines introduced at sites in transmembrane segment S6, which contributes to the intracellular entrance. Within the intracellular half of S6 we found an abrupt cessation of protection for both large and small blockers that is inconsistent with the narrow 'inner pore' seen in the KcsA structure. These and other results are most readily explained by supposing that the structure of Kv channels differs from that of the non-voltage-gated bacterial channel by the introduction of a sharp bend in the inner (S6) helices. This bend would occur at a Pro-X-Pro sequence that is highly conserved in Kv channels, near the site of activation gating.

We have investigated the possible implication of the cell cycle-regulated K(+) channel ether à go-go (EAG) in cell proliferation and transformation. We show that transfection of EAG into mammalian cells confers a transformed phenotype. In addition, human EAG mRNA is detected in several somatic cancer cell lines, despite being preferentially expressed in brain among normal tissues. Inhibition of EAG expression in several of these cancer cell lines causes a significant reduction of cell proliferation. Moreover, the expression of EAG favours tumour progression when transfected cells are injected into immune-depressed mice. These data provide evidence for the oncogenic potential of EAG.

Reduction of an inwardly rectifying K+ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical activity increases in GH3 rat anterior pituitary cells. However, the existence of an inwardly rectifying K+ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target in pituitary cells, including GH3 cells. In this report, an inwardly rectifying component of K+ current is indeed demonstrated in perforated-patch voltage-clamped GH3 cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active. Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current. Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K+ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is probably mediated by a HERG-like K+ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production. Thus, an inwardly-rectifying K+ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH3 cells.

Treatment of rat anterior pituitary GH3 cells with caffeine causes a reversible enhancement of electrical activity superimposed over a depolarization of the plasma membrane potential. Similar results are obtained with theophylline, but not with isobutylmethylxanthine or forskolin. The effects of caffeine are not related to Ca2+ liberation from intracellular stores since they are not affected by incubation of the cells with ryanodine or thapsigargin. Furthermore, caffeine-induced hyperpolarization of the membrane is not detectable even in cells in which Ca2+ liberation from inositol 1,4,5-trisphosphate-sensitive compartments produces a prominent transient hyperpolarization in response to thyrotropin-releasing hormone. Reductions of Ca2+-dependent K+ currents caused by partial block of L-type Ca2+ channels by caffeine are not sufficient to explain the effects of the xanthine, since the results obtained with caffeine are not mimicked by direct blockade of Ca2+ channels with nisoldipine. GH3 cell inwardly rectifying K+ currents are inhibited by caffeine. Studies on the voltage dependence of the caffeine-induced effects indicate a close correlation between alterations of electrical parameters and reported values of steady-state voltage dependence of inactivation of these currents. We conclude that, as previously shown for thyrotropin-releasing hormone, modulation of inwardly rectifying K+ currents plays a major role determining the firing rate of GH3 cells and its enhancement by caffeine.

Exposure of GH3 rat anterior pituitary cells to cholera toxin for 2-4 h significantly increased the thyrotropin-releasing-hormone(TRH)-induced inhibition of the inwardly rectifying K+ current studied in patch-perforated voltage-clamped cells. On the other hand, the current reduction became almost totally irreversible after washout of the neuropeptide. Comparison of the effects elicited by the toxin with those of 8-(4-chlorophenylthio)-cAMP or forskolin plus isobutylmethylxanthine indicated that, although the irreversibility may be due, at least in part, to elevations of cAMP levels, the enhancement of the TRH-induced inhibition of the current is not mediated by the cyclic nucleotide. Only reductions on the inwardly rectifying K+ current, but not those elicited by TRH on voltage-dependent Ca2+ currents, were increased by the treatment with cholera toxin. In current-clamped cells showing similar rates of firing, the second phase of enhanced action-potential frequency induced by TRH was also significantly potentiated by cholera toxin. Measurements of [Ca2+]i oscillations associated with electrical activity, using video imaging with fura-2-loaded cells, demonstrated that cholera toxin treatment causes a clear reduction of spontaneous [Ca2+]i oscillations. However, this did not prevent the stimulatory effect of TRH on oscillations due to the action potentials. In cholera-toxin-treated cells, the steady-state, voltage dependence of inactivation of the inward rectifier was shifted by nearly 20 mV to more negative values. These data suggest that the inwardly rectifying K+ current plays an important role in maintenance of the resting K+ conductance in GH3 cells. Furthermore, the TRH-induced reductions on this current may be an important factor contributing to the increased cell excitability promoted by the neuropeptide.

Thyrotropin-releasing hormone (TRH) reduces an inwardly rectifying K+ current in whole-cell voltage-clamped GH3 rat anterior pituitary cells. The TRH effect depends on the maintenance of a background level of Ca2+ in the pipette buffer, and is rapidly minimized by the intracellular dialysis produced under whole-cell conditions. Introduction of ADP-NH-P, a non-hydrolizable ATP analog, in the pipettes, nearly abolishes the TRH-evoked inhibition. The TRH-induced reduction of the inwardly rectifying current is significantly enhanced by incubation of cells 2-4 h with cholera toxin, but not by inclusion of 1 mM cyclic AMP in the pipettes. Under control whole-cell conditions, the reduction caused by TRH is not reversed upon washout of the neuropeptide. However, this effect is readily reversed by addition of purified catalytic subunits of protein phosphatase 2A (PP-2Ac) but not PP-1c to the buffer used to fill the patch pipettes. Among previous results with PP inhibitors, these data indicate that PP2A is involved in the phosphorylation/dephosphorylation mechanism(s) that regulate the delayed TRH effects on GH3 cell excitability.