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Modulation of human erg K+ channel gating by activation of a G protein‐coupled receptor and protein kinase C

1 Modulation of the human ether‐à‐go‐go‐related gene (HERG) K+ channel was studied in two‐electrode voltage‐clamped Xenopus oocytes co‐expressing the channel protein and the thyrotropin‐releasing hormone (TRH) receptor. 2 Addition of TRH caused clear modifications of HERG channel gating kinetics. These variations consisted of an acceleration of deactivation, as shown by a faster decay of hyperpolarization‐induced tail currents, and a slower time course of activation, measured using an envelope of tails protocol. The voltage dependence for activation was also shifted by nearly 20 mV in the depolarizing direction. Neither the inactivation nor the inactivation recovery rates were altered by TRH. 3 The alterations in activation gating parameters induced by TRH were demonstrated in a direct way by looking at the increased outward K+ currents elicited in extracellular solutions in which K+ was replaced by Cs+. 4 The effects of TRH were mimicked by direct pharmacological activation of protein kinase C (PKC) with β‐phorbol 12‐myristate, 13‐acetate (PMA). The TRH‐induced effects were antagonized by GF109203X, a highly specific inhibitor of PKC that also abolished the PMA‐dependent regulation of the channels. 5 It is concluded that a PKC‐dependent pathway links G protein‐coupled receptors that activate phospholipase C to modulation of HERG channel gating. This provides a mechanism for the physiological regulation of cardiac function by phospholipase C‐activating receptors, and for modulation of adenohypophysial neurosecretion in response to TRH.

Differential effects of amino-terminal distal and proximal domains in the regulation of human erg K(+) channel gating.

The participation of amino-terminal domains in human ether-a-go-go (eag)-related gene (HERG) K(+) channel gating was studied using deleted channel variants expressed in Xenopus oocytes. Selective deletion of the HERG-specific sequence (HERG Delta138-373) located between the conserved initial amino terminus (the eag or PAS domain) and the first transmembrane helix accelerates channel activation and shifts its voltage dependence to hyperpolarized values. However, deactivation time constants from fully activated states and channel inactivation remain almost unaltered after the deletion. The deletion effects are equally manifested in channel variants lacking inactivation. The characteristics of constructs lacking only about half of the HERG-specific domain (Delta223-373) or a short stretch of 19 residues (Delta355-373) suggest that the role of this domain is not related exclusively to its length, but also to the presence of specific sequences near the channel core. Deletion-induced effects are partially reversed by the additional elimination of the eag domain. Thus the particular combination of HERG-specific and eag domains determines two important HERG features: the slow activation essential for neuronal spike-frequency adaptation and maintenance of the cardiac action potential plateau, and the slow deactivation contributing to HERG inward rectification.

Demonstration of an inwardly rectifying K+ current component modulated by thyrotropin-releasing hormone and caffeine in GH3 rat anterior pituitary cells

Abstract 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.

Characteristics and modulation by thyrotropin-releasing hormone of an inwardly rectifying K+ current in patch-perforated GH3 anterior pituitary cells.

Hyperpolarization of patch-perforated GH3 rat anterior pituitary cells in high-K+ Ca2+-free medium reveals an inwardly rectifying K+ current. This current showed potential-dependent activation and inactivation kinetics, complete inactivation during strong hyperpolarization and rectification at depolarized potentials. The current was blocked by millimolar concentrations of external Cs+, Ba2+, Cd2+ and Co2+, but it was almost insensitive to tetraethylammonium, 4-aminopyridine and two dihydropyridines, nisoldipine and nitrendipine. Verapamil and methoxyverapamil produced a strong and reversible inhibition of the current. In the presence of 100 nM thyrotropin-releasing hormone (TRH), the current was reduced. This reduction was increased by holding the cell at more negative potentials and was accompanied by a shift in steady-state voltage dependence of inactivation towards more positive voltages. Furthermore, the current slowly returned to the initial levels upon washout. Treatment of the cell with the protein phosphatase inhibitor okadaic acid increased the magnitude of the inhibition caused by TRH. Moreover, the current did not return towards the control level during a 30-min washout period. It is concluded that protein phosphatases participate in modulation of the GH3 cell inwardly rectifying K+ channels by TRH. Furthermore, these data indicate that either a protein phosphatase or a factor necessary for its activation is lost under whole-cell mode, which could account for the permanent reduction of the current in response to TRH.

The role of the inwardly rectifying K+ current in resting potential and thyrotropin-releasing-hormone-induced changes in cell excitability of GH3 rat anterior pituitary cells.

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 patchperforated 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.

Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains

Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4–S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4–S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.

Protein phosphatase 2A reverses inhibiton of inward rectifying K+ currents by thyrotropin-releasing hormone in GH3 pituitary cells

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.

Thermodynamic and Kinetic Properties of Amino-Terminal and S4-S5 Loop HERG Channel Mutants under Steady-State Conditions

Gating kinetics and underlying thermodynamic properties of human ether-a-go-go-related gene (HERG) K(+) channels expressed in Xenopus oocytes were studied using protocols able to yield true steady-state kinetic parameters. Channel mutants lacking the initial 16 residues of the amino terminus before the conserved eag/PAS region showed significant positive shifts in activation voltage dependence associated with a reduction of z(g) values and a less negative DeltaG(o), indicating a deletion-induced displacement of the equilibrium toward the closed state. Conversely, a negative shift and an increased DeltaG(o), indicative of closed-state destabilization, were observed in channels lacking the amino-terminal proximal domain. Furthermore, accelerated activation and deactivation kinetics were observed in these constructs when differences in driving force were considered, suggesting that the presence of distal and proximal amino-terminal segments contributes in wild-type channels to specific chemical interactions that raise the energy barrier for activation. Steady-state characteristics of some single point mutants in the intracellular loop linking S4 and S5 helices revealed a striking parallelism between the effects of these mutations and those of the amino-terminal modifications. Our data indicate that in addition to the recognized influence of the initial amino-terminus region on HERG deactivation, this cytoplasmic region also affects activation behavior. The data also suggest that not only a slow movement of the voltage sensor itself but also delaying its functional coupling to the activation gate by some cytoplasmic structures possibly acting on the S4-S5 loop may contribute to the atypically slow gating of HERG.

Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered.

Molecular Determinants of Interactions between the N-Terminal Domain and the Transmembrane Core That Modulate hERG K+ Channel Gating

A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4–S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET) analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF) conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4–S5 linker.