Christiane K. Bauer

Modulation of rat erg1, erg2, erg3 and HERG K+ currents by thyrotropin-releasing hormone in anterior pituitary cells via the native signal cascade.

1 The mechanism of thyrotropin‐releasing hormone (TRH)‐induced ether‐à‐go‐go‐related gene (erg) K+ current modulation was investigated with the perforated‐patch whole‐cell technique in clonal somatomammotroph GH3/B6 cells. These cells express a small endogenous erg current known to be reduced by TRH. GH3/B6 cells were injected with cDNA coding for rat erg1, erg2, erg3 and HERG K+ channels. The corresponding erg currents were isolated with the help of the specific erg channel blockers E‐4031 and dofetilide and their biophysical properties were determined. 2 TRH (1 μm) was able to significantly reduce the different erg currents. The voltage dependence of activation was shifted by 15 mV (erg1), 10 mV (erg2) and 6 mV (erg3) to more positive potentials without strongly affecting erg inactivation. TRH reduced the maximal available erg current amplitude by 12 % (erg1), 13 % (erg2) and 39 % (erg3) and accelerated the time course of erg1 and erg2 channel deactivation, whereas erg3 deactivation kinetics were not significantly altered. The effects of TRH on HERG currents did not differ from those on its rat homologue erg1. In addition, coinjection of rat MiRP1 with HERG cDNA did not influence the TRH‐induced modulation of HERG channels. 3 Rat erg1 currents recorded in the cell‐attached configuration were reduced by application of TRH to the extra‐patch membrane in the majority of the experiments, confirming the involvement of a diffusible second messenger. 4 Application of the phorbol ester phorbol 12‐myristate 13‐acetate (PMA; 1 μm) shifted the voltage dependence of erg1 activation in the depolarizing direction, but it did not reduce the maximal current amplitude. The voltage shift could not be explained by a selective effect on protein kinase C (PKC) since the PKC inhibitor bisindolylmaleimide I did not block the effects of TRH and PMA on erg1. In addition, cholecystokinin, known to activate the phosphoinositol pathway similarly to TRH, did not significantly affect the erg1 current. 5 Various agents interfering with different known TRH‐elicited cellular responses were not able to completely mimic or inhibit the TRH effects on erg1. Tested substances included modulators of the cAMP‐protein kinase A pathway, arachidonic acid, inhibitors of tyrosine kinase and mitogen‐activated protein kinase, sodium nitroprusside and cytochalasin D. 6 The results demonstrate that all three members of the erg channel subfamily are modulated by TRH in GH3/B6 cells. In agreement with previous studies on the TRH‐induced modulation of the endogenous erg current in prolactin‐secreting anterior pituitary cells, the TRH effects on overexpressed erg1 channels are not mediated by any of the tested signalling pathways.

The erg‐like potassium current in rat lactotrophs

1 The ether‐à‐go‐go‐related gene (erg)‐like K+ current in rat lactotrophs from primary culture was characterized and compared with that in clonal rat pituitary cells (GH3/B6). The class III antiarrhythmic E‐4031 known to block specifically erg K+ channels was used to isolate the erg‐like current as the E‐4031‐sensitive current. The experiments were performed in 150 mM K+ external solution using the patch‐clamp technique. 2 The erg‐like K+ current elicited with hyperpolarizing pulses negative to ‐100 mV consisted of a fast and a pronounced slowly deactivating current component. The contribution of the slow component to the total current amplitude was potential dependent and varied from cell to cell. At ‐100 mV it ranged from 50 to 85 % and at ‐140 mV from 21 to 45 %. 3 The potential‐dependent channel availability curves determined with 2 s prepulses were fitted with the sum of two Boltzmann functions. The function related to the slowly deactivating component of the erg‐like current was shifted by more than 40 mV to more negative membrane potentials compared with that of the fast component. 4 In contrast to that of native lactotrophs studied under identical conditions, the erg‐like K+ current of GH3/B6 cells was characterized by a predominant fast deactivating current component, with similar kinetic and steady‐state properties to the fast deactivating current component of native lactotrophs. 5 Thyrotrophin‐releasing hormone reduced the erg‐like current in native lactotrophs via an intracellular signal cascade which seemed to involve a pathway independent from protein kinase A and protein kinase C. 6 RT‐PCR studies on cytoplasm from single lactotrophs revealed the presence of mRNA of the rat homologue of the human ether‐à‐go‐go‐related gene HERG (r‐erg1) as well as mRNA of the two other cloned r‐erg cDNAs (r‐erg2 and r‐erg3) in different combinations. In GH3/B6 cells, only the transcripts of r‐erg1 and r‐erg2 were found.

Functions of erg K+ channels in excitable cells

Ether‐à‐go‐go‐related gene (erg) channels are voltage‐dependent K+ channels mediating inward‐rectifying K+ currents because of their peculiar gating kinetics. These characteristics are essential for repolarization of the cardiac action potential. Inherited and acquired malfunctioning of erg channels may lead to the long QT‐syndrome. However, erg currents have also been recorded in many other excitable cells, like smooth muscle fibres of the gastrointestinal tract, neuroblastoma cells or neuroendocrine cells. In these cells erg currents contribute to the maintenance of the resting potential. Changes in the resting potential are related to cell‐specific functions like increase in hormone secretion, frequency adaptation or increase in contractility.

Functional Characterization of Genetically Labeled Gonadotropes

Gonadotropes are crucial in the control of reproduction but difficult to isolate for functional analysis due to their scattered distribution in the anterior pituitary gland. We devised a binary genetic approach, and describe a new mouse model that allows visualization and manipulation of gonadotrope cells. Using gene targeting in embryonic stem cells, we generated mice in which Cre recombinase is coexpressed with the GnRH receptor, which is expressed in gonadotrope cells. We show that we can direct Cre-mediated recombination of a yellow fluorescent protein reporter allele specifically in gonadotropes within the anterior pituitary of these knock-in mice. More than 99% of gonadotropin-containing cells were labeled by yellow fluorescent protein fluorescence and readily identifiable in dissociated pituitary cell culture, allowing potentially unbiased sampling from the gonadotrope population. Using electrophysiology, calcium imaging, and the study of secretion on the single-cell level, the functional properties of gonadotropes isolated from male mice were analyzed. Our studies demonstrate a significant heterogeneity in the resting properties of gonadotropes and their responses to GnRH. About 50% of gonadotropes do not exhibit secretion of LH or FSH. Application of GnRH induced a broad range of both electrophysiological responses and increases in the intracellular calcium concentration. Our mouse model will also be able to direct expression of other Cre recombination-dependent reporter genes to gonadotropes and, therefore, represents a versatile new tool in the understanding of gonadotrope biology.

Mutations in KCNH1 and ATP6V1B2 cause Zimmermann-Laband syndrome

Zimmermann-Laband syndrome (ZLS) is a developmental disorder characterized by facial dysmorphism with gingival enlargement, intellectual disability, hypoplasia or aplasia of nails and terminal phalanges, and hypertrichosis. We report that heterozygous missense mutations in KCNH1 account for a considerable proportion of ZLS. KCNH1 encodes the voltage-gated K+ channel Eag1 (Kv10.1). Patch-clamp recordings showed strong negative shifts in voltage-dependent activation for all but one KCNH1 channel mutant (Gly469Arg). Coexpression of Gly469Arg with wild-type KCNH1 resulted in heterotetrameric channels with reduced conductance at positive potentials but pronounced conductance at negative potentials. These data support a gain-of-function effect for all ZLS-associated KCNH1 mutants. We also identified a recurrent de novo missense change in ATP6V1B2, encoding the B2 subunit of the multimeric vacuolar H+ ATPase, in two individuals with ZLS. Structural analysis predicts a perturbing effect of the mutation on complex assembly. Our findings demonstrate that KCNH1 mutations cause ZLS and document genetic heterogeneity for this disorder.

A FUNCTIONAL ROLE OF THE ERG-LIKE INWARD-RECTIFYING K+ CURRENT IN PROLACTIN SECRETION FROM RAT LACTOTROPHS

The functional role of the inward-rectifying erg-like K+ current in rat lactotrophs was studied by the use of a selective blocker, the class III antiarrhythmic agent E-4031. The erg-like current was measured as drug-sensitive current in physiological K+ gradient. In the range of the normal resting membrane potential of rat lactotrophs (around -45 mV) the erg-like current constituted a steady outward current. A selective block of this current by E-4031 resulted in a moderate (5 mV) depolarization of the membrane potential in 64% of the lactotroph cells. Measurements of basal prolactin secretion with the reverse hemolytic plaque assay showed that the number of prolactin secreting cells and the amount of prolactin secreted from single lactotrophs was significantly increased in the presence of E-4031. The data show that the contribution of the erg-like K+ current to the maintenance of the resting membrane potential is functionally important for the regulation of prolactin secretion.

Erg1, erg2 and erg3 K channel subunits are able to form heteromultimers

Abstract. Clonal somato-mammotroph GH3/B6 cells and lactotroph MMQ cells express two (erg1, erg2) of the three cloned rat ether-à-go-go-related gene (erg) K channel subunits. To study whether the erg subunits form heteromultimers, dominant-negative mutants of erg1 and erg2 were constructed by point mutation (erg1G630S, erg2G480S). After co-expression of these mutants with the wild-type erg1, erg2, or erg3 in Chinese hamster ovary (CHO) cells no erg currents could be detected. In contrast, in co-expression experiments with members of the other ether-à-go-go (EAG) subfamilies (eag1, elk1) the mutant erg1G630S had no effect. These results strongly suggest that erg channel subunits are able to form heteromultimers within the erg channel subfamily. Suppression of the endogenous E-4031-sensitive currents in GH3/B6 and MMQ cells by erg1G630S confirms that they are mediated by erg channels despite the differences in gating kinetics in these cells. Reduction of the erg current in GH3/B6 cells by erg2G480S indicates that erg heteromultimers can also be formed in these cells.

The erg inwardly rectifying K+ current and its modulation by thyrotrophin-releasing hormone in giant clonal rat anterior pituitary cells.

1 The voltage‐dependent inwardly rectifying K+ current (IK,IR) of clonal rat anterior pituitary cells (GH3/B6) was investigated in solutions with physiological K+ gradient using giant polynuclear cells. 2 I K,IR was isolated by the use of the selective erg (ether‐à‐go‐go‐related gene) channel blocker E‐4031. In external 5 mM K+ solution, IK,IR carried steady‐state outward current in the potential range between –60 and 0 mV, with a maximum current amplitude at –40 mV. Negative to the K+ equilibrium potential, EK, large transient inward currents occurred. 3 A selective pharmacological block of IK,IR induced a sustained depolarization of the membrane potential when Ca2+ action potentials were blocked, confirming the contribution of IK,IR to the resting membrane potential of GH3/B6 cells. 4 Thyrotrophin‐releasing hormone (TRH) reduced effectively the sustained outward and the transient inward IK,IR. The magnitude of a TRH‐induced depolarization of the membrane potential was consistent with an almost complete reduction of IK,IR. 5 The results demonstrate that the TRH‐induced reduction of IK,IR is able to mediate the resting potential depolarization, suggesting that the increase in the frequency of action potentials occurring during the second phase of the TRH response in GH cells should be sustained by IK,IR inhibition. Moreover, this is the first evidence of a ligand‐induced physiological modulation of an erg‐mediated current.

The class III antiarrhythmic agent E-4031 selectively blocks the inactivating inward-rectifying potassium current in rat anterior pituitary tumor cells (GH3/B6 cells)

Abstract Hyperpolarization-elicited potassium currents in GH3/B6 cells bathed in high-potassium external solution were recorded to assess effects of the class III antiarrhythmic agent E-4031 on the inactivating inward-rectifying potassium current (IK,IR). E-4031 potently blocked IK,IR with an IC50 value of 10 nM. The complete block of IK,IR achieved with concentrations ≥ 1 μM revealed the presence of a non-inactivating outward-rectifying current which contributed to the membrane currents recorded under control conditions. The time dependence of the IK,IR block depended on the concentration of E-4031. Two other methanesulfonanilides were investigated: WAY-123,398 (10 μM) also totally blocked IK,IR, while sotalol (100 μM) was almost ineffective. Also lanthanum (100 μM) had only a very small effect on IK,IR. E-4031 did not affect sodium, calcium and voltage-dependent outward-rectifying potassium currents, suggesting a selective block of IK,IR in GH3/B6 cells. In an external solution containing 16 mM potassium, the E-4031-sensitive current was present as a steady outward current within a broad potential range positive to the potassium equilibrium potential, EK. In many, but not all, cells E-4031 induced an increase in the frequency of action potentials suggesting an important role of IK,IR in controlling cell excitability. Our experiments show that E-4031 is a valuable tool in characterizing IK,IR and its physiological function.

Biophysical properties of heteromultimeric erg K+ channels

Abstract. The three ether-à-go-go-related gene (erg) K+ channel subunits are able to form heteromultimers within their subfamily. The functional importance of this finding is indicated by in situ hybridization experiments showing that the different erg subunits have overlapping expression patterns in several regions of the brain. To investigate the biophysical properties of heteromultimeric erg channels, concatemers of two erg subunits were constructed and expressed heterologously in Chinese hamster ovary (CHO) cells. The resulting currents were measured using the patch-clamp technique. The heteromultimers exhibited an intermediate potential dependence of activation compared with the corresponding wild-type (WT) erg channels. In contrast, the time course of activation was clearly dominated by the faster activating subunit. The kinetics of recovery from inactivation and the deactivation kinetics of all heteromultimers were similar to those of WT erg1 channels, the rat homologue of the human erg1 K+ channel (HERG), even if erg1 was not part of the concatemer. Taken together, the biophysical properties of heteromultimeric erg channels result in larger current amplitudes upon both depolarization and repolarization. Thus, through heteromeric assembly erg channels may contribute significantly to different physiological functions such as setting and stabilizing the resting membrane potential and modulation of action potential frequency.