Ion Channel Function and Electrical Excitability in the Zona Glomerulosa: A Network Perspective on Aldosterone Regulation.

Aldosterone excess is a pathogenic factor in many hypertensive disorders. The discovery of numerous somatic and germline mutations in ion channels in primary hyperaldosteronism underscores the importance of plasma membrane conductances in determining the activation state of zona glomerulosa (zG) cells. Electrophysiological recordings describe an electrically quiescent behavior for dispersed zG cells. Yet, emerging data indicate that in native rosette structures in situ, zG cells are electrically excitable, generating slow periodic voltage spikes and coordinated bursts of Ca2+ oscillations. We revisit data to understand how a multitude of conductances may underlie voltage/Ca2+ oscillations, recognizing that zG layer self-renewal and cell heterogeneity may complicate this task. We review recent data to understand rosette architecture and apply maxims derived from computational network modeling to understand rosette function. The challenge going forward is to uncover how the rosette orchestrates the behavior of a functional network of conditional oscillators to control zG layer performance and aldosterone secretion.

Pannexin 1 channels in renin-expressing cells influence renin secretion and blood pressure homeostasis.

Kidney function and blood pressure homeostasis are regulated by purinergic signaling mechanisms. These autocrine/paracrine signaling pathways are initiated by the release of cellular ATP, which influences kidney hemodynamics and steady-state renin secretion from juxtaglomerular cells. However, the mechanism responsible for ATP release that supports tonic inputs to juxtaglomerular cells and regulates renin secretion remains unclear. Pannexin 1 (Panx1) channels localize to both afferent arterioles and juxtaglomerular cells and provide a transmembrane conduit for ATP release and ion permeability in the kidney and the vasculature. We hypothesized that Panx1 channels in renin-expressing cells regulate renin secretion in vivo. Using a renin cell-specific Panx1 knockout model, we found that male Panx1 deficient mice exhibiting a heightened activation of the renin-angiotensin-aldosterone system have markedly increased plasma renin and aldosterone concentrations, and elevated mean arterial pressure with altered peripheral hemodynamics. Following ovariectomy, female mice mirrored the male phenotype. Furthermore, constitutive Panx1 channel activity was observed in As4.1 renin-secreting cells, whereby Panx1 knockdown reduced extracellular ATP accumulation, lowered basal intracellular calcium concentrations and recapitulated a hyper-secretory renin phenotype. Moreover, in response to stress stimuli that lower blood pressure, Panx1-deficient mice exhibited aberrant "renin recruitment" as evidenced by reactivation of renin expression in pre-glomerular arteriolar smooth muscle cells. Thus, renin-cell Panx1 channels suppress renin secretion and influence adaptive renin responses when blood pressure homeostasis is threatened.

Role of voltage-gated calcium channels in the regulation of aldosterone production from zona glomerulosa cells of the adrenal cortex.

Zona glomerulosa cells (ZG) of the adrenal gland constantly integrate fluctuating ionic, hormonal and paracrine signals to control the synthesis and secretion of aldosterone. These signals modulate Ca2+ levels, which provide the critical second messenger to drive steroid hormone production. Angiotensin II is a hormone known to modulate the activity of voltage-dependent L- and T-type Ca2+ channels that are expressed on the plasma membrane of ZG cells in many species. Because the ZG cell maintains a resting membrane voltage of approximately -85 mV and has been considered electrically silent, low voltage-activated T-type Ca2+ channels are assumed to provide the primary Ca2+ signal that drives aldosterone production. However, this view has recently been challenged by human genetic studies identifying somatic gain-of-function mutations in L-type CaV 1.3 channels in aldosterone-producing adenomas of patients with primary hyperaldosteronism. We provide a review of these assumptions and challenges, and update our understanding of the state of the ZG cell in a layer in which native cellular associations are preserved. This updated view of Ca2+ signalling in ZG cells provides a unifying mechanism that explains how transiently activating CaV 3.2 channels can generate a significant and recurring Ca2+ signal, and how CaV 1.3 channels may contribute to the Ca2+ signal that drives aldosterone production.

Non-canonical Wnt signaling triggered by WNT2B drives adrenal aldosterone production.

The steroid hormone aldosterone, produced by the zona glomerulosa (zG) of the adrenal gland, is a master regulator of plasma electrolytes and blood pressure. While aldosterone control by the renin-angiotensin system is well understood, other key regulatory factors have remained elusive. Here, we replicated a prior association between a non-coding variant in WNT2B and an increased risk of primary aldosteronism, a prevalent and debilitating disease caused by excessive aldosterone production. We further show that in both mice and humans, WNT2B is expressed in the mesenchymal capsule surrounding the adrenal cortex, in close proximity to the zG. Global loss of Wnt2b in the mouse results in a dysmorphic and hypocellular zG, with impaired aldosterone production. Similarly, humans harboring WNT2B loss-of-function mutations develop a novel form of Familial Hyperreninemic Hypoaldosteronism, designated here as Type 4. Additionally, we demonstrate that WNT2B signals by activating the non-canonical Wnt/planar cell polarity pathway. Our findings identify WNT2B as a key regulator of zG function and aldosterone production with important clinical implications.

Intrinsic Adrenal TWIK-Related Acid-Sensitive TASK Channel Dysfunction Produces Spontaneous Calcium Oscillations Sufficient to Drive AngII (Angiotensin II)-Unresponsive Hyperaldosteronism.

BACKGROUND: Ion channel mutations in calcium regulating genes strongly associate with AngII (angiotensin II)-independent aldosterone production. Here, we used an established mouse model of in vivo aldosterone autonomy, Cyp11b2-driven deletion of TWIK-related acid-sensitive potassium channels (TASK-1 and TASK-3, termed zona glomerulosa [zG]-TASK-loss-of-function), and selective pharmacological TASK channel inhibition to determine whether channel dysfunction in native, electrically excitable zG cell rosette-assemblies: (1) produces spontaneous calcium oscillatory activity and (2) is sufficient to drive substantial aldosterone autonomy. METHODS: We imaged calcium activity in adrenal slices expressing a zG-specific calcium reporter (GCaMP3), an in vitro experimental approach that preserves the native rosette assembly and removes potentially confounding extra-adrenal contributions. In parallel experiments, we measured acute aldosterone production from adrenal slice cultures. RESULTS: Absent from untreated WT slices, we find that either adrenal-specific genetic deletion or acute pharmacological TASK channel inhibition produces spontaneous oscillatory bursting behavior and steroidogenic activity (2.4-fold) that are robust, sustained, and equivalent to activities evoked by 3 nM AngII in WT slices. Moreover, spontaneous activity in zG-TASK-loss-of-function slices and inhibitor-evoked activity in WT slices are unresponsive to AngII regulation over a wide range of concentrations (50 pM to 3 µM). CONCLUSIONS: We provide proof of principle that spontaneous activity of zG cells within classic rosette assemblies evoked solely by a change in an intrinsic, dominant resting-state conductance can be a significant source of AngII-independent aldosterone production from native tissue.

TASK channel deletion in mice causes primary hyperaldosteronism.

When inappropriate for salt status, the mineralocorticoid aldosterone induces cardiac and renal injury. Autonomous overproduction of aldosterone from the adrenal zona glomerulosa (ZG) is also the most frequent cause of secondary hypertension. Yet, the etiology of nontumorigenic primary hyperaldosteronism caused by bilateral idiopathic hyperaldosteronism remains unknown. Here, we show that genetic deletion of TWIK-related acid-sensitive K (TASK)-1 and TASK-3 channels removes an important background K current that results in a marked depolarization of ZG cell membrane potential. Although TASK channel deletion mice (TASK-/-) adjust urinary Na excretion and aldosterone production to match Na intake, they produce more aldosterone than control mice across the range of Na intake. Overproduction of aldosterone is not the result of enhanced activity of the renin-angiotensin system because circulating renin concentrations remain either unchanged or lower than those of control mice at each level of Na intake. In addition, TASK-/- mice fail to suppress aldosterone production in response to dietary Na loading. Autonomous aldosterone production is also demonstrated by the failure of an angiotensin type 1 receptor blocker, candesartan, to normalize aldosterone production to control levels in TASK-/- mice. Thus, TASK-/- channel knockout mice exhibit the hallmarks of primary hyperaldosteronism. Our studies establish an animal model of nontumorigenic primary hyperaldosteronism and identify TASK channels as a possible therapeutic target for primary hyperaldosteronism.

Rosette morphology in zona glomerulosa formation and function.

How morphology informs function is a fundamental biological question. Here, we review the morphological features of the adrenal zona glomerulosa (zG), highlighting recent cellular and molecular discoveries that govern its formation. The zG consists of glomeruli enwrapped in a Laminin-ß1-enriched basement membrane (BM). Within each glomerulus, zG cells are organized as rosettes, a multicellular structure widely used throughout development to mediate epithelial remodeling, but not often found in healthy adult tissues. Rosettes arise by constriction at a common cellular contact point mediated/facilitated by adherens junctions (AJs). In mice, small, dispersed AJs first appear postnatally and enrich along the entire cell-cell contact around 10 days after birth. Subsequently, these AJ-rich contacts contract, allowing rosettes to form. Concurrently, flat sheet-like domains in the nascent zG, undergo invagination and folding, gradually giving rise to the compact round glomeruli that comprise the adult zG. How these structures impact adrenal function is discussed.

ATP and large signaling metabolites flux through caspase-activated Pannexin 1 channels.

Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.

T-type calcium channel regulation by specific G-protein betagamma subunits.

Low-voltage-activated (LVA) T-type calcium channels have a wide tissue distribution and have well-documented roles in the control of action potential burst generation and hormone secretion. In neurons of the central nervous system and secretory cells of the adrenal and pituitary, LVA channels are inhibited by activation of G-protein-coupled receptors that generate membrane-delimited signals, yet these signals have not been identified. Here we show that the inhibition of alpha1H (Ca(v)3.2), but not alpha(1G) (Ca(v)3.1) LVA Ca2+ channels is mediated selectively by beta2gamma2 subunits that bind to the intracellular loop connecting channel transmembrane domains II and III. This region of the alpha1H channel is crucial for inhibition, because its replacement abrogates inhibition and its transfer to non-modulated alpha1G channels confers beta2gamma2-dependent inhibition. betagamma reduces channel activity independent of voltage, a mechanism distinct from the established betagamma-dependent inhibition of non-L-type high-voltage-activated channels of the Ca(v)2 family. These studies identify the alpha1H channel as a new effector for G-protein betagamma subunits, and highlight the selective signalling roles available for particular betagamma combinations.

L- and T-type calcium channels control aldosterone production from human adrenals.

Aldosterone, which plays a key role in the regulation of blood pressure, is produced by zona glomerulosa (ZG) cells of the adrenal cortex. Exaggerated overproduction of aldosterone from ZG cells causes primary hyperaldosteronism. In ZG cells, calcium entry through voltage-gated calcium channels plays a central role in the regulation of aldosterone secretion. Previous studies in animal adrenals and human adrenal adrenocortical cell lines suggest that the T-type but not the L-type calcium channel activity drives aldosterone production. However, recent clinical studies show that somatic mutations in L-type calcium channels are the second most prevalent cause of aldosterone-producing adenoma. Our objective was to define the roles of T and L-type calcium channels in regulating aldosterone secretion from human adrenals. We find that human adrenal ZG cells mainly express T-type CaV3.2/3.3 and L-type CaV1.2/1.3 calcium channels. TTA-P2, a specific inhibitor of T-type calcium channel subtypes, reduced basal aldosterone secretion from acutely prepared slices of human adrenals. Surprisingly, nifedipine, the prototypic inhibitor of L-type calcium channels, also decreased basal aldosterone secretion, suggesting that L-type calcium channels are active under basal conditions. In addition, TTA-P2 or nifedipine also inhibited aldosterone secretion stimulated by angiotensin II- or elevations in extracellular K+. Remarkably, blockade of either L- or T-type calcium channels inhibits basal and stimulated aldosterone production to a similar extent. Low concentrations of TTA-P2 and nifedipine showed additive inhibitory effect on aldosterone secretion. We conclude that T- and L-type calcium channels play equally important roles in controlling aldosterone production from human adrenals.

Glycosylation of CaV3.2 Channels Contributes to the Hyperalgesia in Peripheral Neuropathy of Type 1 Diabetes.

Our previous studies implicated glycosylation of the CaV3.2 isoform of T-type Ca2+ channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant CaV3.2 channel by de-glycosylation enzymes such as neuraminidase (NEU) and PNGase-F (PNG), as well as their behavioral and biochemical effects in painful PDN Type 1. In our in vitro study we used whole-cell recordings of current-voltage relationships to confirm that CaV3.2 current densities were decreased ~2-fold after de-glycosylation. Furthermore, de-glycosylation induced a significant depolarizing shift in the steady-state relationships for activation and inactivation while producing little effects on the kinetics of current deactivation and recovery from inactivation. PDN was induced in vivo by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and CaV3.2 knock-out (KO mice). Either NEU or vehicle (saline) were locally injected into the right hind paws or intrathecally. We found that injections of NEU, but not vehicle, completely reversed thermal and mechanical hyperalgesia in diabetic WT rats and mice. In contrast, NEU did not alter baseline thermal and mechanical sensitivity in the CaV3.2 KO mice which also failed to develop painful PDN. Finally, we used biochemical methods with gel-shift analysis to directly demonstrate that N-terminal fragments of native CaV3.2 channels in the dorsal root ganglia (DRG) are glycosylated in both healthy and diabetic animals. Our results demonstrate that in sensory neurons glycosylation-induced alterations in CaV3.2 channels in vivo directly enhance diabetic hyperalgesia, and that glycosylation inhibitors can be used to ameliorate painful symptoms in Type 1 diabetes. We expect that our studies may lead to a better understanding of the molecular mechanisms underlying painful PDN in an effort to facilitate the discovery of novel treatments for this intractable disease.

Angiotensin II induces coordinated calcium bursts in aldosterone-producing adrenal rosettes.

Aldosterone-producing zona glomerulosa (zG) cells of the adrenal gland arrange in distinct multi-cellular rosettes that provide a structural framework for adrenal cortex morphogenesis and plasticity. Whether this cyto-architecture also plays functional roles in signaling remains unexplored. To determine if structure informs function, we generated mice with zG-specific expression of GCaMP3 and imaged zG cells within their native rosette structure. Here we demonstrate that within the rosette, angiotensin II evokes periodic Cav3-dependent calcium events that form bursts that are stereotypic in form. Our data reveal a critical role for angiotensin II in regulating burst occurrence, and a multifunctional role for the rosette structure in activity-prolongation and coordination. Combined our data define the calcium burst as the fundamental unit of zG layer activity evoked by angiotensin II and highlight a novel role for the rosette as a facilitator of cell communication.

ß-Catenin and FGFR2 regulate postnatal rosette-based adrenocortical morphogenesis.

Rosettes are widely used in epithelial morphogenesis during embryonic development and organogenesis. However, their role in postnatal development and adult tissue maintenance remains largely unknown. Here, we show zona glomerulosa cells in the adult adrenal cortex organize into rosettes through adherens junction-mediated constriction, and that rosette formation underlies the maturation of adrenal glomerular structure postnatally. Using genetic mouse models, we show loss of ß-catenin results in disrupted adherens junctions, reduced rosette number, and dysmorphic glomeruli, whereas ß-catenin stabilization leads to increased adherens junction abundance, more rosettes, and glomerular expansion. Furthermore, we uncover numerous known regulators of epithelial morphogenesis enriched in ß-catenin-stabilized adrenals. Among these genes, we show Fgfr2 is required for adrenal rosette formation by regulating adherens junction abundance and aggregation. Together, our data provide an example of rosette-mediated postnatal tissue morphogenesis and a framework for studying the role of rosettes in adult zona glomerulosa tissue maintenance and function.

Beta-Catenin Causes Adrenal Hyperplasia by Blocking Zonal Transdifferentiation.

Activating mutations in the canonical Wnt/ß-catenin pathway are key drivers of hyperplasia, the gateway for tumor development. In a wide range of tissues, this occurs primarily through enhanced effects on cellular proliferation. Whether additional mechanisms contribute to ß-catenin-driven hyperplasia remains unknown. The adrenal cortex is an ideal system in which to explore this question, as it undergoes hyperplasia following somatic ß-catenin gain-of-function (ßcat-GOF) mutations. Targeting ßcat-GOF to zona Glomerulosa (zG) cells leads to a progressive hyperplastic expansion in the absence of increased proliferation. Instead, we find that hyperplasia results from a functional block in the ability of zG cells to transdifferentiate into zona Fasciculata (zF) cells. Mechanistically, zG cells demonstrate an upregulation of Pde2a, an inhibitor of zF-specific cAMP/PKA signaling. Hyperplasia is further exacerbated by trophic factor stimulation leading to organomegaly. Together, these data indicate that ß-catenin drives adrenal hyperplasia through both proliferation-dependent and -independent mechanisms.

Emerging roles for two-pore-domain potassium channels and their potential therapeutic impact.

A distinct gene family of widely distributed and well-modulated two-pore-domain background potassium (K(2P)) channels establish resting membrane potential and cell excitability. By using new mouse models in which K(2P)-channel genes are deleted, the contributions of these channels to important physiological functions are now being revealed. Here, we highlight results of recent studies using mice deleted for K(2P)-channel subunits that uncover physiological functions of these channels, mostly those of the TASK and TREK subgroup. Consistent with activation of these K(2P) channels by volatile anesthetics, TASK-1, TASK-3 and TREK-1 contribute to anesthetic-induced hypnosis and immobilization. The acid-sensitive TASK channels are not required for brainstem control of breathing by CO(2) or pH, despite widespread expression in respiratory-related neurons. TASK channels are necessary, however, for homeostatic regulation of adrenal aldosterone secretion. The heat-, stretch- and lipid-activated TREK-1 channels contribute to temperature and mechanical pain sensation, neuroprotection by polyunsaturated fatty acids and, unexpectedly, mood regulation. The alkaline-activated TASK-2 channel is necessary for HCO(3)(-) reabsorption and osmotic volume regulation in kidney proximal tubule cells. Development of compounds that selectively modulate K(2P) channels is crucial for verifying these results and assessing the efficacy of therapies targeting these interesting channels.

Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+/calmodulin-dependent protein kinase II.

Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II-elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with alpha1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression.

Adrenal Tissue-Specific Deletion of TASK Channels Causes Aldosterone-Driven Angiotensin II-Independent Hypertension.

The renin-angiotensin system tightly controls aldosterone synthesis. Dysregulation is evident in hypertension (primary aldosteronism), low renin, and resistant hypertension) but also can exist in normotension. Whether chronic, mild aldosterone autonomy can elicit hypertension remains untested. Previously, we reported that global genetic deletion of 2 pore-domain TWIK-relative acid-sensitive potassium channels, TASK-1 and TASK-3, from mice produces striking aldosterone excess, low renin, and hypertension. Here, we deleted TASK-1 and TASK-3 channels selectively from zona glomerulosa cells and generated a model of mild aldosterone autonomy with attendant hypertension that is aldosterone-driven and Ang II (angiotensin II)-independent. This study shows that a zona glomerulosa-specific channel defect can produce mild autonomous hyperaldosteronism sufficient to cause chronic blood pressure elevation.

Reducing agents sensitize C-type nociceptors by relieving high-affinity zinc inhibition of T-type calcium channels.

Recent studies have demonstrated an important role for T-type Ca2+ channels (T-channels) in controlling the excitability of peripheral pain-sensing neurons (nociceptors). However, the molecular mechanisms underlying the functions of T-channels in nociceptors are poorly understood. Here, we demonstrate that reducing agents as well as endogenous metal chelators sensitize C-type dorsal root ganglion nociceptors by chelating Zn2+ ions off specific extracellular histidine residues on Ca(v)3.2 T-channels, thus relieving tonic channel inhibition, enhancing Ca(v)3.2 currents, and lowering the threshold for nociceptor excitability in vitro and in vivo. Collectively, these findings describe a novel mechanism of nociceptor sensitization and firmly establish reducing agents, as well as Zn2+, Zn2+-chelating amino acids, and Zn2+-chelating proteins as endogenous modulators of Ca(v)3.2 and nociceptor excitability.

Molecular mechanisms of subtype-specific inhibition of neuronal T-type calcium channels by ascorbate.

T-type Ca2+ channels (T-channels) are involved in the control of neuronal excitability and their gating can be modulated by a variety of redox agents. Ascorbate is an endogenous redox agent that can function as both an anti- and pro-oxidant. Here, we show that ascorbate selectively inhibits native Ca(v)3.2 T-channels in peripheral and central neurons, as well as recombinant Ca(v)3.2 channels heterologously expressed in human embryonic kidney 293 cells, by initiating the metal-catalyzed oxidation of a specific, metal-binding histidine residue in domain 1 of the channel. Our biophysical experiments indicate that ascorbate reduces the availability of Ca(v)3.2 channels over a wide range of membrane potentials, and inhibits Ca(v)3.2-dependent low-threshold-Ca2+ spikes as well as burst-firing in reticular thalamic neurons at physiologically relevant concentrations. This study represents the first mechanistic demonstration of ion channel modulation by ascorbate, and suggests that ascorbate may function as an endogenous modulator of neuronal excitability.

A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies.

The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. We developed a family of ACh sensors (GACh) based on G-protein-coupled receptors that has the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal, and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing-pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.