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, 103 (13), 5197-201

Rapid Signaling at the Plasma Membrane by a Nuclear Receptor for Thyroid Hormone

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Rapid Signaling at the Plasma Membrane by a Nuclear Receptor for Thyroid Hormone

Nina M Storey et al. Proc Natl Acad Sci U S A.

Abstract

Many nuclear hormones have physiological effects that are too rapid to be explained by changes in gene expression and are often attributed to unidentified or novel G protein-coupled receptors. Thyroid hormone is essential for normal human brain development, but the molecular mechanisms responsible for its effects remain to be identified. Here, we present direct molecular evidence for potassium channel stimulation in a rat pituitary cell line (GH(4)C(1)) by a nuclear receptor for thyroid hormone, TRbeta, acting rapidly at the plasma membrane through phosphatidylinositol 3-kinase (PI3K) to slow the deactivation of KCNH2 channels already in the membrane. Signaling was disrupted by heterologous expression of TRbeta receptors with mutations in the ligand-binding domain that are associated with neurological disorders in humans, but not by mutations that disrupt DNA binding. More importantly, PI3K-dependent signaling was reconstituted in cell-free patches of membrane from CHO cells by heterologous expression of human KCNH2 channels and TRbeta, but not TRalpha, receptors. TRbeta signaling through PI3K provides a molecular explanation for the essential role of thyroid hormone in human brain development and adult lipid metabolism.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Rapid stimulation of KCNH2 currents by T3 in GH4C1 cells. (A) Voltage protocol and resulting currents elicited from a GH4C1 cell. Four current traces from the same cell are superimposed: control (in blue); perfusion control (in black); 5 min after application of 100 nM T3 (in red); and 8 min after 5 μM E4031 (in green), a selective KCNH2 inhibitor, which completely blocks the T3-stimulated current at −120 mV. (B) Time course of changes in KCNH2 current amplitude measured by voltage protocol repeated at 10-s intervals. (C) Percentage increase in KCNH2 current in the presence of 3 μM 1-850, an antagonist of T3 binding to TRβ, or after overnight transfection with mutant TRβ receptors.
Fig. 2.
Fig. 2.
T3 stimulates individual KCNH2 channels already gating in cell-attached patches of plasma membrane on GH4C1 cell voltage-clamped to 0 mV in high potassium. (A) Voltage protocol repeated at 10-s intervals and 9 representative traces (out of 100) from the same patch of a single KCNH2 channel: 3 traces under control conditions, 3 traces 2 min after addition of 100 nM T3, and 3 traces 2 min after addition of 5 μM E4031. (B) Channel open-time histograms at −60 mV for control and thyroid hormone, T3, treated patches, and the open probability (PO) is indicated. (C) Summary of changes in seven such patches plotting percentage of traces, or “episodes” (100 per patch) where no activity was visible (zero activation) or where activity was sustained for more than half the duration of the voltage-step (long activation).
Fig. 3.
Fig. 3.
T3 stimulates KCNH2 channels in GH4C1 cells through PI3K. (A) Immunoprecipitation of native TRβ from GH4C1 cell lysates pulls down native p85, but this association is reduced after 5-min treatment with 100 nM T3. (B) Immunoprecipitation of native p85 from CHO cell lysates pulls down heterologously expressed GFP-TRβ1, and this association is also reduced after 5 min in 100 nM T3. (C) Confocal images of GH4C1 cells illustrate the effect of 100 nM T3 for 15 min on lamellipodia [differential interference contrast microscopy (DIC)], Rac distribution (green), and F-actin organization (red). Pretreatment with 50 nM wortmannin prevents the T3-induced changes. (D) Whole-cell currents elicited by the illustrated family of voltage steps from a control cell (Upper) and from a cell transfected overnight with p85 (Lower). (E) Single channel recording of KCNH2 activity in cell-free patch. Control records and response to 5 μM PIP3.
Fig. 4.
Fig. 4.
KCNH2 stimulation by T3 is reconstituted in CHO cells with recombinant TRβ2. (A) Voltage-protocol and representative currents from a CHO cell expressing human KCNH2 and TRβ2. T3 (100 nM, red) increases control current (black), and 5 μM E4031 blocks all of the current at −120 mV. (B) Histograms comparing current stimulated by T3 in cells expressing TRβ2 or TRα1. (C) Voltage protocol and representative single-channel currents elicited from a cell-free patch taken from a CHO cell expressing KCNH2 and TRβ2. Ensemble averages of 10 traces before and after 100 nM T3 are displayed. (D) Histograms showing the percentage increase in ensemble channel activity produced by 100 nM T3 in cells transfected with TRβ2 WT or GS125.
Fig. 5.
Fig. 5.
TRβ signaling in the nucleus through DNA binding or in the cytoplasm through PI3K can lead to changes in gene expression. Production of PIP3 also stimulates cell metabolism through the PIP3-dependent protein kinase (PDK) and cytoskeleton through guanine exchange factors for the Rac GTPase. The mechanism of KCNH2 stimulation by Rac is addressed in a separate article (37).

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