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

Abstract

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 Ca_V1.3 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 Ca_V3.2 channels can generate a significant and recurring Ca²⁺ signal, and how Ca_V1.3 channels may contribute to the Ca²⁺ signal that drives aldosterone production.

Abbreviations

APA

aldosterone producing adenoma

CDI

Ca²⁺‐dependent inactivation

IHA

idiopathic bilateral zona glomerulosa hyperplasia

PA

primary hyperaldosteronism

RAS

renin–angiotensin system

ZG

zona glomerulosa

Primary hyperaldosteronism

Circulating aldosterone is synthesized and secreted by zona glomerulosa (ZG) cells of the adrenal cortex. When under the control of the renin–angiotensin system (RAS), aldosterone production is commensurate with the level of circulating renin. By promoting Na⁺ retention at multiple sites along the nephron, aldosterone contributes to the physiological regulation of electrolytes and water balance. Excessive aldosterone production independent of the RAS can occur, as in primary hyperaldosteronism (PA), and is characterized by a large increase in the aldosterone/renin ratio (Montori & Young, 2002). Because aldosterone overproduction in PA has additional adverse effects on the heart (Catena et al. 2008) and kidneys (Rossi et al. 2006) that are independent of blood pressure elevation, this most common type of endocrine hypertension is associated with increased cardiovascular risk (Stowasser, 2001; Savard et al. 2013). The most common forms of PA result from two major pathologies: unilateral aldosterone producing adenoma (APA) and idiopathic bilateral zona glomerulosa hyperplasia (IHA). With a prevalence of only 4% in the general population, the incidence of PA rises significantly among hypertensive patients (> 10%) (Plouin et al. 2004; Rossi et al. 2006) and markedly among those with resistant hypertension (20%) (Calhoun et al. 2002 a,b; Stowasser, 2014).

Pathogenic mechanisms: insights from human genetic analysis of channels and pumps

Since 2011, great progress has been made in our understanding of the genetic bases for APA. Choi et al. (2011) were the first group to identify recurrent somatic mutations in the coding region of the KCNJ5 G‐protein coupled, inwardly rectifying K⁺ channel in DNA extracted from aldosterone producing adenomas. These mutations occur in or near the selectivity filter of the channel rendering it permeable to Na⁺. Originally identified in a small cohort of 22 severely hypertensive patients, these or functionally equivalent mutations in exon 2 of the KCNJ5 gene are now the most frequently identified genetic variants (∼38%) in aldosterone‐producing adenomas (Azizan et al. 2012; Boulkroun et al. 2012; Mulatero et al. 2012; Dekkers et al. 2014; Fernandes‐Rosa et al. 2014; Kuppusamy et al. 2014). Less frequent variants have been identified in the P‐type ATPase gene family, ATP1A1 and ATP2B3, encoding Na⁺/K⁺‐ATPase and Ca²⁺‐ATPase3, respectively (∼7%) (Azizan et al. 2013; Beuschlein et al. 2013; Fernandes‐Rosa et al. 2014). Mutations residing in transmembrane helices M1 and M4 of the Na⁺/K⁺‐ATPase cause loss of functional pump activity, the appearance of new ouabain‐insensitive Na⁺ or H⁺ inward leak currents and cell depolarization (Azizan et al. 2013; Beuschlein et al. 2013). Cell depolarization is also induced by mutations in the Ca²⁺‐ATPase3 found in the M4 transmembrane helix that forms the Ca²⁺ binding pocket of the Ca²⁺ pump (Beuschlein et al. 2013). By contrast, multiple and more frequent (5–14%) somatic mutations have been identified in the CACNA1D gene encoding the Ca_V1.3 voltage‐dependent Ca²⁺ channel (Azizan et al. 2013; Scholl et al. 2013; Fernandes‐Rosa et al. 2014). These variants are scattered broadly throughout the large α1 subunit of the channel protein, and are found in the activation gate (S6 helices), the voltage sensor (S4 helices) and in the S4‐S5 cytoplasmic linkers connecting the voltage sensor to the channel pore. In general, these mutations result in a gain of Ca²⁺ channel function, increasing the probability for channel opening at hyperpolarized potentials, either alone or in combination with a shift in the voltage dependence of channel inactivation. Some mutations also dramatically slow the time dependence of inactivation. In addition, several germline mutations in the KCNJ5 gene outside the selectivity filter were recently described in patients with apparent sporadic PA (Murthy et al. 2014), and a rare recurrent mutation in the CACNA1H gene encoding the Ca_V3.2 voltage‐dependent Ca²⁺ channel was identified in children presenting with severe hyperaldosteronism and hypertension (Scholl et al. 2015). The latter mutation found in the IIIS6 domain markedly slows the time dependence of inactivation of this rapidly inactivating channel without altering the voltage dependencies of channel gating.

Interestingly, the mutational status of tumours among different patient cohorts does not support the hypothesis that APA is associated with a consistent histological phenotype; mutations do not segregate according to cell type (zona glomerulosa, zona fasciculata or mixed). Neither was there a consistent molecular signature (e.g. the overexpression of CYP11B2, KCNK5, CACNA1D, ATP1A1 genes) (Enberg et al. 2004; Lenzini et al. 2007; Fernandes‐Rosa et al. 2014; Boulkroun et al. 2015). One potential exception may include the CACNA1H gene whose level of mRNA expression significantly associates with peripheral blood aldosterone levels, CYP11B2 gene expression levels and KCNJ5 mutational status in a cohort of 74 Japanese APA patients (Felizola et al. 2014). A second exception may involve the KCNK5 gene whose mRNA expression level inversely correlates with aldosterone synthesis and microRNA expression in a subcohort of 32 confirmed APA patients of Italian origin (Rossi et al. 2006 a; Lenzini et al. 2014). While the universality of these findings awaits their replication in multiethnic cohorts, the data in aggregate indicate that APA is a disorder with highly variable cellular and molecular phenotypes with diverse genetic underpinnings.

Despite the lack of consistent genotype–phenotype correlations (Boulkroun et al. 2015; Zennaro et al. 2015), the bulk of evidence supports the hypothesis that independent, mutational‐based mechanisms can support a persistent elevation in Ca²⁺ that drives the overproduction of aldosterone. Specifically, all of these mutations – directly or indirectly – are expected to raise intracellular Ca²⁺ in ZG cells, either by an increase in Ca²⁺ channel open probability (Ca_V1.3, Ca_V3.2), by a loss of PMCA3 Ca²⁺ pump activity, or by a depolarization in cell membrane potential, an action predicted to reduce Na⁺–Ca²⁺ exchange activity and/or activate voltage‐dependent Ca²⁺ channels.

Calcium sites of action and regulation of the Ca²⁺ signal

In the ZG cell, Ca²⁺ is the critical second messenger that regulates the production of aldosterone. The steroidogenic, intracellular Ca²⁺ signal is generated by the opening of Ca²⁺‐selective, voltage‐dependent channels found at the plasma membrane, or by the release of Ca²⁺ from intracellular stores (Fakunding & Catt, 1980; Kojima et al. 1984; Barrett et al. 1989; Rasmussen et al. 1989). The subsequent increase in cytosolic Ca²⁺ facilitates the delivery of cholesterol to the mitochondria (Cherradi et al. 1996) for its conversion to pregnenolone, a precursor in the aldosterone biosynthetic pathway. To persistently increase steroidogenesis, the Ca²⁺ signal must also be transferred to the mitochondrial matrix (Lalevee et al. 2003) where it stimulates matrix dehydrogenases to generate NADH. NADH, in turn, is converted to NADPH, a cofactor that is required for two critical regulatory steps in the biosynthetic pathway: (1) the conversion of cholesterol to pregnenolone catalysed by CYP11A1, and (2) the conversion of deoxycorticosterone to aldosterone catalysed by CYP11B2 (Rossier et al. 1996; Wiederkehr et al. 2011). Thus, a Ca²⁺ increase in both the cytosolic and the mitochondrial compartments of the ZG cell is required to increase the production of aldosterone that persists for minutes to hours (Spat & Hunyady, 2004).

Voltage‐gated calcium currents

Early electrophysiological recordings in rat, bovine and human ZG cells consistently identified two components of the Ca²⁺ current that could be attributed to distinct Ca²⁺ channel classes based on voltage‐dependent gating properties, kinetics and pharmacology (Matsunaga et al. 1987; Cohen et al. 1988; Durroux et al. 1988; Payet et al. 1994). Typically, the more persistent current that was elicited by strong depolarization (from resting V _m of −85 mV to > −50 mV (Ca_V1.3), or > −30 mV (Ca_V1.2)) (Xu & Lipscombe, 2001) and inhibited by dihydropyridines characterized the high voltage‐activated component (HVA). By contrast, the rapid, transient current that was evoked by weak depolarization (from resting V _m to > −65 mV) and inhibited by Ni²⁺ characterized the low voltage‐activated component (LVA) (Tsien et al. 1988). Later, the molecular correlates of these currents were identified as HVA, L‐type (i.e. Ca_V1.3 and Ca_V1.2) and LVA, T‐type (i.e. Ca_V3.2 and Ca_V3.1) channels. The relative mRNA abundance of L‐ and T‐type channel isoforms in adrenal ZG varies among studies and species (Lesouhaitier et al. 2001; Schrier et al. 2001; Rossier et al. 2003) although their biophysical properties remain similar. Additionally, functional N‐type channels (Ca_V2.0) have been recorded in isolated rat ZG cells (Durroux et al. 1988). Thus, the repertoire of Ca²⁺ channels found in ZG cells is rich.

More recently, the expression profiles for voltage‐gated Ca²⁺ channels in normal and pathological human adrenocortical tissue were compared (Scholl et al. 2013; Felizola et al. 2014). mRNA for all three Ca²⁺ channel classes was detected in human aldosterone‐producing adenomas, with greater relative abundance of Ca_V1.3 and Ca_V3.2 mRNA than Ca_V1.2 and Ca_V2.2 mRNA (Felizola et al. 2014). Surprisingly, the protein expression of Ca_V1.3 and Ca_V3.2 channels is consistent regardless of tissue health, as anti‐Ca_V1.3 or anti‐Ca_V3.2 immunoreactivity is equivalent in APA, IHA and normal adrenocortical tissue samples. However, whether all detected protein is functional remains a critical unanswered question.

Calcium homeostasis: lessons learned from isolated cells

From the late 1970s to early 1990s, methods to dissociate and isolate ZG cells from bovine and rat adrenal tissues led to tractable preparations suitable for measuring ZG cell Ca²⁺/K⁺ currents, intracellular Ca²⁺ signals and aldosterone production. Numerous studies document that the major regulators of aldosterone production, angiotensin II and extracellular K⁺, raise intracellular Ca²⁺ in ZG cells (Capponi et al. 1984, 1987; Connor et al. 1987; Kramer, 1988; Johnson et al. 1989; Pratt et al. 1989; Rossig et al. 1996) and that changes in cell Ca²⁺ are commensurate with a striking sustained depolarization of the ZG cell. Angiotensin II elicits ZG cell depolarization by inhibiting hyperpolarizing conductances, predominantly those mediated by leak and voltage‐gated K⁺ channels (Quinn et al. 1987 b; Brauneis et al. 1991; Lotshaw, 1997 a; Guagliardo et al. 2012), whereas elevated extracellular K⁺ induces a shift in the K⁺ equilibrium potential across the highly K⁺‐conductive ZG plasma membrane (Quinn et al. 1987 a,b; Lotshaw, 1997 b). As depolarization recruits the activation of voltage‐gated Ca²⁺ channels, these early findings suggested the possibility that voltage‐gated Ca²⁺ entry is an important contributor to ZG cell activation and aldosterone production.

With the subsequent development of pharmacological agents to reduce the open‐state probability of selective Ca²⁺ channel classes, as well as hormonal and molecular approaches to alter Ca²⁺ channel gating mechanisms, the relative contribution of T‐type and L‐type Ca²⁺ currents to the regulation of aldosterone production was confirmed. Given the extremely hyperpolarized resting membrane potential of isolated ZG cells (∼−85 mV), the relatively modest ∼5–20 mV depolarizing shifts in voltage evoked by physiological concentrations of angiotensin ll or K⁺, coupled with robust T‐type channel currents recorded in all preparations, it is not surprising that T‐type and not L‐type Ca²⁺ current amplitude (Barrett et al. 1995; Rossier et al. 1996; Lotshaw, 2001) strongly correlated with aldosterone output in these studies (data from 9 previous studies compiled and reviewed by Rossier (2006)). Thus, voltage‐gated Ca²⁺ channels have assumed a privileged role in ZG cell Ca²⁺ homeostasis.

Framed by the aforementioned and other studies, the ZG cell came to be regarded as electrically quiescent, operating over a narrow, relatively hyperpolarized voltage range in which the open probability of T‐type Ca²⁺ channels is low, but also in which the steady‐state inactivation of the channel is incomplete. The latter, it was argued, promoted a small‐yet‐discernible Ca²⁺ conductance into the ZG cell via a T channel ‘window current’ that was functionally sufficient to sustain the production of aldosterone (Cohen et al. 1988; Lotshaw, 2001; Wolfe et al. 2002). However, at odds with this description of an electrically silent ZG cell were several findings: (1) angiotensin II elicited oscillatory changes in cytosolic free Ca²⁺ in a small percentage of isolated cells that persisted for several minutes before transitioning to a tonic elevation (Johnson et al. 1989); (2) oscillatory [Ca²⁺]_i responses preferentially evoked by low concentrations of angiotensin II (Quinn et al. 1988) depended only on voltage‐gated Ca²⁺ entry and not IP₃‐induced Ca²⁺ release (Rossig et al. 1996); (3) spontaneous Ca²⁺‐dependent voltage spike potentials were recorded in ZG cells retained within cat adrenal slices (Natke & Kabela, 1979); and (4) spike potentials in isolated ZG cells could be evoked by depolarizing current injection or by pharmacological blockade of K⁺ currents (Quinn et al. 1987). More recently, our laboratory extended these findings and discovered that ZG cells are indeed electrically excitable when their cellular connectivity is preserved within a tissue slice (Hu et al. 2012). Specifically, ZG cells organized within cortical rosette structures spontaneously generate periodic (∼0.5 Hz), large depolarizing amplitude changes (∆ +75 mV from −85 mV) in membrane potential (V _m oscillations) that are modulated in frequency by angiotensin II and extracellular K⁺. Collectively, these observations demonstrate that Ca²⁺ levels within ZG cells are highly dynamic and, therefore, motivated a further evaluation of the excitability of the ZG cell.

Pacemakers in the zona glomerulosa

Many excitable cells, ranging from those found in invertebrate (Lewis, 1988; Lamb & Calabrese, 2012; Marder et al. 2015) to mammalian (Steriade et al. 1993; Marcantoni et al. 2010; Vandael et al. 2010) systems, have the capacity to generate electrical oscillations, even when isolated. In many cases, these intrinsic oscillations are driven by a small ensemble of distinct ion channels that operate in concert to generate periodic changes in voltage. These so‐called pacemaker channel conductances often rely on voltage‐gated Ca²⁺ channels to produce the large, rising phase that characterizes the depolarizing component of the voltage oscillation. For example, a well‐characterized oscillator reliant upon T‐type Ca²⁺ channels is found in thalamocortical neurons of the thalamus, in which an interplay between T‐type Ca²⁺ channels and hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels drives voltage oscillations likely to be important for memory consolidation during sleep (Gais et al. 2002; Eschenko et al. 2006; Fogel et al. 2011). A second, well‐characterized oscillator reliant upon voltage‐gated Ca²⁺ channels is in the catecholamine secreting adrenal chromaffin cell in which there is an interplay between Ca_V1.3 channels and Ca²⁺‐activated K⁺ channels (BK, SK). In this cell, the relatively low threshold for activation of Ca_V1.3 currents at interspike potentials (∼−50 mV) drives depolarization and Ca²⁺ entry which is sufficient to activate BK channels within restricted Ca²⁺ domains and highly Ca²⁺‐sensitive SK channels at more distal sites. Once active these K⁺ channels hyperpolarize the membrane, remove Ca_V1.3 channel inactivation and enable the initiation of a new oscillatory cycle (Marcantoni et al. 2010; Vandael et al. 2010; Vandael & Carbone, 2015).

The recurrent observation that biological pacemakers often recruit voltage‐gated Ca²⁺ channels to produce oscillations motivated our laboratory to identify ion channels that endow the ZG cell with the capacity to operate as a pacemaker. While such identification is incomplete, we have learned that targeting Na_V1.x channels with TTX or Ca_V1.x channels with nifedipine fails to alter either the amplitude or the frequency of ZG V _m oscillations. In contrast, low concentrations of Ni²⁺ that selectively target Ca_V3.x channels halt V _m oscillations, indicating an important contribution of these low voltage‐activated Ca²⁺ channels to ZG pacemaking (Hu et al. 2012). Considering that Ca_V3.2 is the primary Ca_V3.x member expressed in the ZG layer, we hypothesize that activation of Ca_V3.2 channels underlies the rapidly depolarizing phase of the ZG cell oscillation. The identification of the ion channel(s) that contribute to the falling phase remains less well delineated. However, silencing V _m oscillations with Ni²⁺ does not cause a hyperpolarization as would be predicted from block of T‐type Ca²⁺ current, but rather causes a 20 mV depolarization from baseline (to ∼−60 mV), suggestive of a functional link between Ca_V3.2 and a Ca²⁺‐activated K⁺ current(s). In this model, Ca²⁺ entry through Ca_V3.2 channels both depolarizes the ZG cell and recruits Ca²⁺‐activated K⁺ channels which serve to hyperpolarize the cell; thus, the Ni²⁺‐evoked depolarization likely reflects the closure of the latter. It is notable that the peak amplitude of Ca_V3.2 pacemaker current is during the repolarization phase of the oscillatory cycle; this temporal dependence would allow Ca_V3.2 current to activate Ca²⁺‐activated K⁺ channels to complete the oscillatory cycle. Together these data provide evidence, albeit indirect, that a Ca²⁺‐activated K⁺ conductance may participate in the falling phase (hyperpolarization phase) of the ZG cell oscillation (Fig. 1).

Figure 1. A working model of the ZG pacemaker .

At the start of the oscillatory cycle (V _m = −85 mV), depolarizing conductance(s) (X_v) elicit the opening of low voltage‐activated Ca_v3.2 channels which further depolarize V _m and enable the opening of high voltage‐activated Ca_v1.3 channels. Ca²⁺ influx and a reduced V _m recruits the activity of Ca²⁺‐dependent K⁺ channels that return V _m to −85 mV to begin another oscillatory cycle.

The aforementioned ZG pacemaker model is developed from observations amassed from normal, wild‐type mice. In adapting these findings to human ZG cells, or to ZG cell behaviour during pathological conditions, it is important to consider the potential contribution of L‐type Ca²⁺ channels (Ca_V1.3), the mRNA of which is also found in normal human adrenals, aldosterone‐producing adenomas (Azizan et al. 2013; Scholl et al. 2013; Fernandes‐Rosa et al. 2014) and recently in aldosterone‐producing clusters (Nishimoto et al. 2015) in addition to that for Ca_V3.2. Two salient features of Ca_V1.3 channels are worth noting when considering their contribution to ZG function. First, the voltage range over which Ca_V1.3 channels carry Ca²⁺ current is ∼25 mV more hyperpolarized than prototypical L‐type Ca²⁺ channels, potentially enabling them to be active within the voltage range of the ZG cell oscillation (−85 to −10 mV). Although Ca_V1.3 channels would not be active at rest (−85 mV), they would be recruited in the oscillatory cycle following activation of lower threshold depolarizing conductances, including Ca_V3.2 channels. However, because Ca_V1.3 channels undergo rapid Ca²⁺‐dependent inactivation (CDI) (Xu & Lipscombe, 2001), it remains unclear to what extent these channels contribute to ZG pacemaker function or aldosterone production under non‐pathological conditions. Indeed, it is worth noting that in animal species that express T‐type and L‐type Ca²⁺ channels (rat, bovine), T‐type current amplitude but not L‐type current correlates with aldosterone production (Barrett et al. 1995; Rossier et al. 1996 b; Lotshaw, 2001; Rossier, 2006).

By contrast, it is likely that the contribution of mutant Ca_V1.3 channels to exaggerated aldosterone production is substantial. As discussed above, many gain‐of‐function mutations in the CACNA1D gene encoding Ca_V1.3 channels are observed in aldosterone‐producing adenomas (Azizan et al. 2013; Scholl et al. 2013; Fernandes‐Rosa et al. 2014) and aldosterone producing clusters (Nishimoto et al. 2015). As many of these mutations shift Ca_V1.3 gating properties to substantially more hyperpolarized membrane voltages, the reliance of Ca_v1.3 channel activation on lower threshold conductances would be mitigated, thereby allowing channels to open at rest and also to conduct Ca²⁺ under a greater inward driving force. In principle Ca²⁺ entry could also be augmented by disrupting CDI (Xu & Lipscombe, 2001). Based on structure–function analysis of Ca_V1.3 channels, mutations that alter activation gating but spare voltage‐dependent inactivation (VDI) would be most likely to alter CDI (Tadross et al. 2010). However, to date most mutations in APAs have not been experimentally tested for disrupted CDI and in the one study that has, CDI remained unperturbed (Scholl et al. 2013). Nevertheless, the modulation of this channel gating property of Ca_V1.3 could provide an additional mechanism for regulating Ca²⁺ entry and cellular excitability by altering the rate of spontaneous and evoked oscillations independent of changes in the voltage gating properties of Ca_V1.3 channels (see Fig. 1; Scharinger et al. 2015).

Potential relevance of ZG oscillations

If we accept the new place of the ZG cell among pacemakers, what then is the physiological relevance of ZG voltage oscillations? Do ZG cell voltage oscillations simply exist to recruit a range of voltage‐dependent Ca²⁺ conductances that collectively ensure a significant rise in cytosolic Ca²⁺ such that aldosterone is produced? In such a context, ZG Ca²⁺ oscillations would be epiphenomenal, i.e. a large, non‐periodic increase in cytosolic Ca²⁺ could achieve the same effect. Or do periodic oscillations provide a unique opportunity for Ca²⁺ channels with divergent biophysical properties to subserve singular functions in regulating ZG cell activity and aldosterone production? Support for such a possibility comes directly from ZG studies and also from many more summarized and formalized by McCobb and Beam (1991).

First, as determined by oscillatory voltage‐clamp experiments conducted in ZG cells, ZG V _m oscillations are sufficient in magnitude and correct in frequency to drive repetitive, large‐amplitude Ca_V3.2‐mediated currents known as low threshold calcium spikes. Thus, V _m oscillations in ZG cells provide a mechanism for rapidly inactivating Ca_V3.2 channels to generate long‐lasting, periodic Ca²⁺ signals in ZG cells that would be much larger than those associated with T‐channel window currents reliant upon incomplete channel inactivation (Hu et al. 2012). Second, because T‐type Ca²⁺ channels have the lowest threshold for activation and have a relatively slow rate of closure (deactivation), Ca²⁺ entry through these channels is driven by more negative driving forces. Thus, Ca²⁺ entry can be quite large and is favoured in response to a rapidly repolarizing oscillatory wave‐form (McCobb & Beam, 1991), such as recorded in ZG cells. Third, because L‐type Ca²⁺ channels have a more depolarized inactivation threshold and slower inactivation kinetics, entry through L‐type Ca²⁺ channels would be favoured in response to oscillatory waveforms that decay/repolarize slowly and have extended durations. Thus, as has been elegantly demonstrated and formalized by McCobb and colleagues, because of the high sensitivity of L‐type Ca²⁺ channels to the width of the oscillatory waveform, L‐type Ca²⁺ channels would provide the ZG cell with a mechanism to report activity changes in K⁺ channels that control the rate of repolarization. By contrast, given the relative insensitivity of T‐type Ca²⁺ channels to oscillatory waveform width (McCobb & Beam, 1991), Ca²⁺ entry through T‐type channels would better report ZG oscillation frequency. Thus, although each channel type can convert an electrical signal into a chemical Ca²⁺ response, when presented with an oscillatory electrical signal they do so advantaged by their biophysical properties. Thus, ZG V _m oscillations may allow the cell to functionally compartmentalize the contribution of distinct voltage‐gated Ca²⁺ channels.

Finally, it is important to consider how ZG cells might process a periodic Ca²⁺ signal and how such a signal could control the rate of aldosterone production. In this regard the biochemical and computational studies of Ca²⁺/calmodulin‐dependent protein kinase (CaM kinase II) are noteworthy. In this multimeric enzyme complex, individual subunits of CaMKII are activated by high threshold periodic pulses of Ca²⁺. After the threshold for kinase activation is attained, some subunits are trans‐phosphorylated at Thr²⁸⁶ to generate a persistent, subthreshold kinase activity that is autonomous of the Ca²⁺ signal (Hanson et al. 1989). This positive feedback loop augments responses to low frequency stimuli and allows CaMKII to act as a decoder of the frequency of oscillatory Ca²⁺ signals (De Koninck & Schulman, 1998). In a similar manner, it is formally possible that periodic Ca²⁺ signals within ZG cells are more potent drivers of aldosterone production than static Ca²⁺ signals, because in ZG cells CaMKII activation induces a hyperpolarizing shift in the V _1/2 of activation of T‐type channels (Welsby et al. 2003; Yao et al. 2006). Such an increase in T‐current amplitude could thus provide a greater depolarizing current for L‐type channel recruitment further increasing extracellular Ca²⁺ entry. This possibility awaits future examination.

Final remarks

Here, we provide a brief review of recent mutational analyses performed on human adrenal tissues associated with excessive aldosterone production, and address how the newly defined ZG pacemaker cell, by driving the cell to membrane potentials that support channel activity, may unmask these mutations, many of which are associated with voltage‐dependent ion channels. In attempting to address the latter, we are confronted with many uncertainties. Clearly, to completely understand how a mutated ion channel contributes to excessive steroidogenic signalling, we must first better define the collection of ionic conductances that govern ZG cell excitability in the normal, healthy state. And yet, our current knowledge of the ionic conductances that drive ZG pacemaker activity is primarily limited to how the low threshold, T‐type Ca²⁺ channel (Ca_V3.2) drives ZG membrane potential to depolarized levels. To this end, we must also define all currents that mediate the rapid repolarization phase of the ZG pacemaker oscillation. Only when all the relevant channels comprising the ZG pacemaker are identified and the pacemaker regulation of aldosterone production is known, will we be able to fully understand by what means specific mutations support greater aldosterone production. Clearly, the future remains an exciting time for ion channel physiology within the realm of adrenal function.

Additional information

Competing interests

The authors have no competing interests.

Author contributions

All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Biography

Mark P. Beenhakker received his PhD from University of Pennsylvania and is currently Assistant Professor in the Department of Pharmacology. Currently his group focuses on thalamocortical oscillations in epilepsy. Paula Q. Barrett received her PhD from the University of Rochester and is currently Professor in the Department of Pharmacology. She has had a long standing interest in the ionic regulation of aldosterone production. Nick Guagliardo and Peter Kein, neuroendocrine physiologists, Changlong Hu, an electrophysiologist, and David Breault, a developmental biologist, have provided additional insight. Despite their divergent research foci, their recent work has converged towards the goal of better understanding cellular oscillations in aldosterone producing cells of the adrenal cortex.