Isradipine therapy in Cacna1dIle772Met/+ mice ameliorates primary aldosteronism and neurologic abnormalities

Somatic gain-of-function mutations in the L-type calcium channel CaV1.3 (CACNA1D gene) cause adrenal aldosterone-producing adenomas and micronodules. De novo germline mutations are found in a syndrome of primary aldosteronism, seizures, and neurologic abnormalities (PASNA) as well as in autism spectrum disorder. Using CRISPR/Cas9, we here generated mice with a Cacna1d gain-of-function mutation found in both adenomas and PASNA syndrome (Cacna1dIle772Met/+). These mice show reduced body weight and increased mortality from weaning to approximately 100 days of age. Male mice do not breed, likely due to neuromotor impairment, and the offspring of female mice die perinatally, likely due to lack of maternal care. Mice generated by in vitro fertilization showed elevated intracellular calcium in the aldosterone-producing zona glomerulosa, an elevated aldosterone/renin ratio, and persistently elevated serum aldosterone on a high-salt diet as signs of primary aldosteronism. Anesthesia with ketamine and xylazine induced tonic-clonic seizures. Neurologic abnormalities included hyperlocomotion, impaired performance in the rotarod test, impaired nest building, and slight changes in social behavior. Intracellular calcium in the zona glomerulosa, aldosterone levels, and rotarod performance responded to treatment with the calcium channel blocker isradipine, with implications for the therapy of patients with aldosterone-producing lesions and with PASNA syndrome.


Introduction
Hypertension affects approximately one-third of the adult population (1) and is a major contributor to morbidity and mortality worldwide (2).Most cases are considered primary.A causative underlying disease (secondary hypertension) is found in approximately 10% of patients.Primary aldosteronism (PA), the excessive production of the adrenal steroid hormone aldosterone, is the most common cause of secondary hypertension.PA affects at least 50 million people worldwide (3), but recent studies have found evidence of partially autonomous aldosterone production in > 10% of normotensive individuals and > 20% of patients with hypertension (4).PA can be caused by either aldosterone-producing adenomas (benign tumors), multiple aldosterone-producing micronodules (smaller lesions), or diffuse hyperplasia of the adrenal gland (5).Aldosterone-producing micronodules also occur in healthy individuals (6).More than 95% of aldosterone-producing adenomas and 60%-80% of micronodules from patients with PA without adenoma carry somatic mutations in known disease genes (7).In 2013, we and others discovered heterozygous somatic gain-of-function mutations in the CACNA1D gene, encoding voltage-gated L-type calcium channel Ca V 1.3, as a cause of aldosterone-producing adenomas (8,9).These mutations account for ~20% of tumors in individuals of recent European ancestry, ~40% in those of recent African ancestry, and ~15% in those of Asian ancestry.For unknown reasons, they are more prevalent in men than in women (10).CACNA1D is the most frequently Somatic gain-of-function mutations in the L-type calcium channel Ca V 1.3 (CACNA1D gene) cause adrenal aldosterone-producing adenomas and micronodules.De novo germline mutations are found in a syndrome of primary aldosteronism, seizures, and neurologic abnormalities (PASNA) as well as in autism spectrum disorder.Using CRISPR/Cas9, we here generated mice with a Cacna1d gain-of-function mutation found in both adenomas and PASNA syndrome (Cacna1d Ile772Met/+ ).These mice show reduced body weight and increased mortality from weaning to approximately 100 days of age.Male mice do not breed, likely due to neuromotor impairment, and the offspring of female mice die perinatally, likely due to lack of maternal care.Mice generated by in vitro fertilization showed elevated intracellular calcium in the aldosterone-producing zona glomerulosa, an elevated aldosterone/renin ratio, and persistently elevated serum aldosterone on a high-salt diet as signs of primary aldosteronism.Anesthesia with ketamine and xylazine induced tonic-clonic seizures.Neurologic abnormalities included hyperlocomotion, impaired performance in the rotarod test, impaired nest building, and slight changes in social behavior.Intracellular calcium in the zona glomerulosa, aldosterone levels, and rotarod performance responded to treatment with the calcium channel blocker isradipine, with implications for the therapy of patients with aldosterone-producing lesions and with PASNA syndrome.
Gross phenotype, survival, and breeding of Cacna1d Ile772Met/+ mice.We initially attempted to propagate Cacna1d Ile772Met/+ mice by breeding them with C57BL/6J WT mice.Despite multiple attempts, no pregnancies were achieved when housing male Cacna1d Ile772Met/+ mice with female WT mice, and no plugs were observed.Video recordings demonstrated attempted mountings; however, male Cacna1d Ile772Met/+ mice appeared to lack the strength and/or motor coordination for successful mating (Supplemental Table 1 and Supplemental Videos 1-4; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.162468DS1;observation of 3 male WT and 3 male Cacna1d Ile772Met/+ mice).Breeding of female Cacna1d Ile772Met/+ mice with male WT mice resulted in pregnancies; however, the offspring were found dead shortly after birth.Sperm isolated from a Cacna1d Ile772Met/+ mouse showed normal motility and was used for in vitro fertilization (IVF), which resulted in the birth of viable offspring.In the second generation, conventional breeding was again unsuccessful, and all further experimental animals were generated by IVF.Among animals genotyped at weaning, Cacna1d Ile772Met/+ mice (n = 79) were not underrepresented compared with WT (n = 68), suggesting that perinatal lethality was not increased.However, we observed increased mortality of Cacna1d Ile772Met/+ mice between weaning and approximately 100 days of age (Figure 2A), after which no further lethality was observed.Pathological investigation in one case revealed brownish visceral organs and a sparsely filled gastrointestinal tract, but no specific cause of death was determined.Both male and female Cacna1d Ile772Met/+ mice showed significantly reduced body weight and shorter body length compared with WT littermates (Figure 2, B and C).Organ weights of WT male mice were significantly heavier with the notable exception of brain, with less pronounced effects in female mice (Supplemental Table 2).We next investigated Cacna1d Ile772Met/+ mice for features of PASNA syndrome.
Aldosterone remains elevated on a high-salt diet in Cacna1d Ile772Met/+ mice.To assess whether aldosterone production is -at least partially -uncoupled from regulation by the renin-angiotensin system in Cacna1d Ile- 772Met/+ mice, we fed mice a high-salt diet (4% NaCl in food, 1% NaCl in drinking water) for 2 weeks and measured aldosterone at the end of this period.As expected, aldosterone levels strongly declined in WT mice (32.8 ± 26.6 pg/mL).In Cacna1d Ile772Met/+ mice, levels declined slightly but remained much higher than in WT (Figure 4D; 156.3 ± 83.3 pg/mL).Similarly, adrenal Cyp11b2 levels were significantly elevated in heterozygous mice compared with WT mice after a high-salt diet (Supplemental Figure 1C).Renin concentrations could not be reliably assessed due to the use of isoflurane for anesthesia (30) to prevent induction of seizures (see below), but renal renin expression did not significantly differ between WT and mutant mice after a high-salt diet (Supplemental Figure 1D).
Cacna1d Ile772Met/+ mice show no signs of hyperinsulinemia or hypoglycemia.Serum glucose levels at euthanasia were similar in WT and Cacna1d Ile772Met/+ mice (Table 1).Because isoflurane anesthesia (see below) can increase blood glucose levels but does not affect insulin release (31), we additionally determined serum insulin levels by ELISA.We did not observe significant differences between WT and Cacna1d Ile772Met/+ mice (Supplemental Figure 2).
Cacna1d Ile772Met/+ mice show elevated zona glomerulosa calcium levels.We performed calcium imaging of adrenal slices stained with Fura-2 AM (ratiometric dye, allowing for determination of absolute calcium concentrations).We used variable extracellular concentrations of K + and angiotensin II (Ang II) to model effects of the 2 main stimuli of aldosterone production.The pattern of calcium oscillations was largely unaffected (Figure 5, A and B).Cacna1d Ile772Met/+ mice showed significantly elevated intracellular calcium levels compared with WT across almost all concentrations of Ang II and extracellular K + .They showed the expected increase in calcium signaling in response to increased extracellular K + and supraphysiologic concentration of Ang II (Figure 5C and Table 2).At a concentration of 2 pM Ang II, cells only rarely exhibited calcium spikes, the observed increase in mean calcium therefore reflects an increase in baseline calcium levels (Figure 5C).The higher intracellular calcium levels were not associated with increased frequencies of calcium spiking (Supplemental Figure 3A), and bursting parameters were similar, apart from increased numbers of bursts under some conditions (Supplemental Figure 3B).
Anesthesia with ketamine and xylazine induces tonic-clonic seizures in Cacna1d Ile772Met/+ mice.We did not observe spontaneous seizure activity in Cacna1d Ile772Met/+ mice in video recordings of 4 mice spanning approximately 24 hours each.However, upon i.p. injection of ketamine and xylazine for anesthesia, 10 of 13 Cac-na1d Ile772Met/+ mice showed abnormal jerking movements and tonic-clonic seizures (Supplemental Video 5).

R E S E A R C H A R T I C L E
JCI Insight 2023;8(20):e162468 https://doi.org/10.1172/jci.insight.162468 No seizures were observed in 19 similarly treated WT mice (P < 1 × 10 -5 , Fisher's exact test).We subsequently changed anesthesia to isoflurane, under which we did not observe further seizures.
Cacna1d Ile772Met/+ mice show multiple neurologic abnormalities.We performed a modified SmithKline Beecham, Harwell, Imperial College, and Royal London Hospital phenotype assessment (SHIRPA) test battery as a generalized neurological screen of WT and heterozygous mice (Supplemental Table 3) (32).Besides reduced body weight (Figure 2B), this revealed tremor almost exclusively in Cacna1d Ile772Met/+ mice (36 of 37 in Het versus 2 of 42 in WT; P = 1.9 × 10 -19 , Fisher's exact test) and reduced grip strength and grasping (wire maneuver test; n WT = 42, n Het = 37; median score WT: 0, median Het: 2; Mann-Whitney U, U = 523, P = 3.7 × 10 -10 ).Auditory startle response to a tone from a clicker was intact in both WT and Cacna1d Ile772Met/+ mice.We also did not find evidence of impaired olfaction in a buried food-seeking test (33) (Supplemental Figure 4A), and mice showed normal recognition memory of a familiar object (Supplemental Figure 4B).To further test motor function, we performed the rotarod test.Cacna1d Ile772Met/+ mice showed impaired coordination, with reduced times spent on  6C) (WT: n = 19, 6.4% ± 5.6%; Het: n = 19, 4.9% ± 3.7%).Cacna1d Ile772Met/+ mice showed severely impaired nest building scores (Figure 6D; mean ± SD; WT: n = 19, 2.7 ± 1.3; Het: n = 19, 1.3 ± 0.5), which have been linked to structural or functional brain deficits (34).To further investigate these abnormalities, we performed home cage screening (HCS), in which behavior is automatically categorized based on video recordings of singly housed mice in cages for a period of 24 hours (Supplemental Figure 5).Cacna1d Ile772Met/+ mice showed increased locomotion (Figure 7, A and B) compared with WT mice particularly at the start of the dark period (active period of mice), with decreasing locomotion in the later phase of the dark period.A similar peak in activity was observed after the change to the light phase (Figures 7, A and B).This was not due to a change in resting patterns (Supplemental Figure 5).In line with reduced grip strength observed in the SHIRPA test, Cacna1d Ile772Met/+ mice spent less time hanging at the top of the cage lid (Figure 7C).
Social behavior in Cacna1d Ile772Met/+ mice.We used the 3-chamber social test to detect changes in preference for a social stimulus (unknown mouse) compared with an empty cage.Cacna1d Ile772Met/+ mice showed a marked preference for a novel social stimulus over a novel empty object (small grid cage) (Figure 8A).Similarly, Cac-na1d Ile772Met/+ mice again preferred a novel social stimulus (new unknown mouse) over a known social stimulus (Figure 8B), whereas no significant preference was seen in WT littermates.Exploration of the novel social stimulus is mostly limited to olfaction, manifesting as sniffing.Counting the number of sniffing events at the new unknown mouse relative to the known mouse revealed a similar pattern with no difference for WT (n = 19, known: 28 ± 9 sniffs, unknown: 29 ± 7 sniffs, Mann-Whitney U, U = 146, P = 0.32) but a preference for the novel stimulus in Cacna1d Ile772Met/+ mice (n = 19, known: 36 ± 10 sniffs, unknown: 55 ± 14 sniffs, Mann-Whitney U, U = 51, P = 2 × 10 -4 ).We additionally performed a social proximity test (forced social interaction in a constrained space) to investigate whether Cacna1d Ile772Met/+ mice show evidence of autistic features.Compared with WT mice, Cacna1d Ile772Met/+ mice showed significantly more nose-to-anogenital contacts, jump escape events, uprighting events, crawl-over events, and crawl under-events when in contact with an unknown mouse, but there was no significant increase in nose tip-to-nose tip contacts or nose-to-head contacts (Figure 8, C-I).
Cacna1d Ile772Met/+ mice do not show evidence of structural brain abnormalities.To investigate whether the observed neurological abnormalities were due to structural brain abnormalities, we assessed overall brain morphology using Nissl staining (35) (Supplemental Figure 6).No major abnormalities were detected, and structures such as cortex, hippocampus, midbrain, cerebellum, thalamus, hypothalamus, and striatum appeared unaltered.Analysis of cell density and nucleus size in the striatum, which plays central roles in not only movement planning and execution but also social behavior (36), did not reveal significant differences between the 2 genotypes.Similarly, the pattern of dopaminergic neurons in the midbrain (including the substantia nigra, crucial for voluntary movement; ref. 37) and the expression of the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (Th), appeared unchanged (Supplemental Figure 7).
Incubation with isradipine lowers intracellular calcium levels in zona glomerulosa cells.To assess whether increased intracellular zona glomerulosa calcium is sensitive to inhibition by a clinically approved drug, we incubated slices with the dihydropyridine-class calcium channel blocker isradipine.We used a concentration of 50 nM, which can be reached in mouse plasma following s.c.delivery with osmotic minipumps (28), as well as 300 nM, which, in a heterologous expression system, inhibited virtually all Ca V 1.3 channels (38).Both WT and Cacna1d Ile772Met/+ glomerulosa cells responded with a decrease in mean intracellular Ca 2+ , and levels in treated Cacna1d Ile772Met/+ cells were lowered to approximately the levels seen in untreated WT cells (Figure 9A and Table 3).Notably, isradipine lowered baseline Ca 2+ concentrations exclusively in Cacna1d Ile772Met/+ cells (Figure 9B and Table 3).
Cacna1d Ile772Met/+ mice respond to oral therapy with isradipine.Implantation of osmotic minipumps ( 28) is a stressful invasive procedure.Given the already-increased mortality in Cacna1d Ile772Met/+ mice, we instead chose oral administration of isradipine.Slow-release isradipine is approved for the therapy of hypertension and taken once daily in humans (39).We fed Cacna1d Ile772Met/+ mice and WT controls once daily with 12.5 mg/kg of a slow-release isradipine formulation in sweetened yogurt.Both the treatment and the placebo group (sweetened yogurt only) typically rapidly and voluntarily ingested the offered yogurt; mice that did not ingest the yogurt were excluded from further analysis.After a 2-week treatment phase, we continued treatment while performing behavioral testing to assess effects on neurologic behavior.We performed rotarod testing approximately 4 hours after yogurt or isradipine feeding, at a time when peak plasma levels are reached in humans (40).In WT animals, no difference in rotarod performance in the therapy group versus placebo was observed.Conversely, the time spent on the rod was, on average, 68% higher in Cacna1d Ile772Met/+ mice treated with isradipine than in Cacna1d Ile772Met/+ mice that received  9C), even though absolute performance still did not reach WT levels (Table 4).No effect was seen in the open-field test 18 hours after the last dose (Supplemental Figure 8B), at which time we expected trough plasma levels (40).Similarly, no change in nest building performance was seen (test covered peak as well as trough plasma levels of isradipine) (Supplemental Figure 8C).We assessed aldosterone levels from a terminal blood collection approximately 20 hours after the last dose.Treatment led to a reduction of serum aldosterone relative to untreated controls in Cacna1d Ile772Met/+ but not WT mice (Figure 9D).Renin concentrations could not be reliably assessed due to the use of isoflurane for anesthesia (30) to prevent induction of seizures (see above).

Discussion
The Cacna1d Ile772Met/+ mice we here generated closely model human PASNA syndrome, showing PA, increased seizure susceptibility, and neurologic abnormalities, including motor deficits and slight social impairment.Cacna1d Ile772Met/+ mice had high aldosterone levels that remained elevated (along with elevated Cyp11b2 levels) after a high-salt diet, confirming PA (41).Mechanistically, calcium concentrations in glomerulosa cells of Ile772Met/+ mice were elevated, but the frequency of spiking and bursting parameters were mostly unchanged (Figure 5 and Supplemental Figure 3).Baseline calcium levels were elevated in the virtual absence of spiking (during perfusion of 2 pM of Ang II or during pauses of spiking  at stronger stimulation (Figure 5), consistent with calcium permeability of the mutant channel at hyperpolarized potentials.Like other models of mild PA (42-44), Cacna1d-knockin mice had unsuppressed renin.Stress-induced stimulation of renin release during application of anesthesia and blood draw may act as a confounder (45).Introduction of the germline Ile772Met mutation in mice did not cause macroscopic or microscopic glomerulosa hyperplasia at 14 weeks of age.Similarly, the child with PASNA syndrome in whom the corresponding Ile770Met de novo germline mutation was identified did not show macroscopic adrenal abnormalities by computed tomography (8).Given that the corresponding Ile-770Met mutation has been identified in human aldosterone-producing adenomas (8), factors other than somatic CACNA1D mutations may be required for increased cell mass in human tumors (46).Because knockin mice were fragile and showed increased mortality, we could not perform invasive blood pressure monitoring in Cacna1d Ile772Met/+ mice.Other models with mild PA show moderately elevated blood pressure (42,43,47).Hypoglycemic hyperinsulinism was not reported in the child with germline Ile770Met mutation (8).Likewise, we did not detect hyperinsulinemic hypoglycemia in our mice.However, we cannot exclude transient neonatal hypoglycemia (8,48).

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The requirement for IVF limited the scope of our studies.Video recordings demonstrated several attempts of Cacna1d Ile772Met/+ males to mount females.However, these remained unsuccessful, likely due to their impaired neuromotor phenotype (Supplemental Table 1).We suspect that the offspring of female Cacna1d Ile772Met/+ mice did not survive due to lack of maternal care and/or feeding because animals carried by synchronized WT recipients survived.
The overall neurologic phenotype of Cacna1d Ile772Met/+ mice is likely due to functional alterations rather than structural brain abnormalities (Supplemental Figure 6).Ca V 1.3 channels play an important All statistics were obtained by a likelihood ratio test of linear mixed models.A P < 0.05; B P < 0.01.role in the dopaminergic system (49).Ca V 1.3 is expressed in substantia nigra dopamine neurons that project to the dorsal striatum and are important for voluntary movements (50).Ca 2+ entry via Ca V 1 channels into these neurons can be inhibited by isradipine, leading to a reduction in average and peak intracellular [Ca 2+ ] levels (51).Thus, increased calcium and subsequent dopamine signaling may explain some aspects of the neurological phenotype of Cacna1d Ile772Met/+ mice.Accordingly, functional changes in dopaminergic neurons of the medial substantia nigra and GABAergic medium spiny neurons in the dorsal striatum were found in a related mouse model with gain-of-function Cacna1d mutation (52).How anesthesia with ketamine and xylazine induced seizures remains to be determined.Very few cases of ketamine-induced seizures have been described in humans and in cats (53), and a case of a potentially xylazine-induced seizure in a horse has been reported (54).It is conceivable that spontaneous seizures also occur in Cacna1d Ile772Met/+ mice, and such events could contribute to the increased lethality of the strain.Video recordings over 24 hours did not show such events, but they were performed at age 17-18 weeks, when lethality was no longer increased compared with WT.
Regarding social interactions, we unexpectedly observed an increased preference of Cacna1d Ile772Met/+ mice for unknown mice in the 3-chamber social preference assay (Figure 8).While the absence of the expected preference for unknown mice in WT mice may point to suboptimal assay conditions, general social interaction and recognition of unknown mice is likely not impaired in Cacna1d Ile772Met/+ mice.We note that different versions of this assay have been developed, with discrepancies in phenotypic descriptions of several autism spectrum disorder mouse models (55).Increased nose-to-anogenital, crawl-under, and crawl-over behaviors of Cacna1d Ile772Met/+ mice in the social proximity assay may reflect avoidance of reciprocal frontal orientations, analogous to gaze aversion in humans with autism spectrum disorder (56).Crawl-under behavior and jump escape have been associated with anxiety and defensiveness (56).Hyperlocomotion, observed in open-field and HCS, with resulting increased overall interactions, may override decreased nose tip-to-nose tip behavior that is otherwise characteristic of autism-like behavior in mice (56).Autism-like behavior is not a predominant feature in Cacna1d Ile772Met/+ mice.This feature appears to be more prominent in mice homozygous for a Cacna1d mutation associated with autism in humans (52), demonstrating that the spectrum from disorder to disease associated with different mutations in humans is well reflected in mouse models.Olfaction and audition, which could have affected social behavior, appeared largely unaffected in Cacna1d Ile772Met/+ mice (Supplemental Table 3 and Supplemental Figure 4A).In the SHIRPA test, there were no major neuromotor abnormalities other than impaired grasping in the wire maneuver and tremor (Supplemental Table 3), yet nest construction was severely impaired (Figure 6D).).For all statistical models, genotype was used as fixed and recording as well as time as random factors.All P values are adjusted for 10 comparisons (including the remaining behaviors shown in Supplemental Figure 5) using the FDR method.Data are shown as mean ± 95% CI.
Interestingly, contrary to a published Cacna1d-KO model with deafness, bradycardia (57), and impaired motor performance (58), our Cacna1d -/-mice were not viable.It would be interesting to determine whether alternative splicing (several splice isoforms, including a long and a short isoform, have been reported; ref. 59) or other mechanisms enable residual Ca V 1.3 function in the published model.
The most remarkable result of our study is the partial response of PASNA syndrome features to therapy with isradipine.Only knockin mice showed improved rotarod performance 4 hours after isradipine administration, when peak plasma levels are expected.Furthermore, in knockin mice, intracellular calcium levels in adrenal slices and plasma aldosterone levels declined upon isradipine administration.When analyzing baseline calcium concentrations outside of spiking events, the effect of isradipine was strikingly restricted to Cacna1d Ile772Met/+ mice (Figure 9B), suggesting that published shifts of channel activation and inactivation to more hyperpolarized potentials (8) cause abnormal constitutive calcium influx at or close to the glomerulosa resting membrane potential in Cacna1d Ile772Met/+ mice.This may explain why isradipine reduced aldosterone levels in knockin mice.Given that isradipine is approved for the therapy of hypertension, calcium channel blocker therapy could be a treatment

Methods
Generation and propagation of the Cacna1d I772M/+ mouse model.CRISPR/Cas9-mediated mutagenesis in the Cacna1d gene (Ile772Met) was performed at the Yale Genome Editing Center as described previously (42) using fertilized eggs from C57BL/6J mice.See Supplemental Methods for more information.
High-salt diet.We fed mice with food pellets containing 1.71% Na + (4% NaCl; EF15431-347, ssniff Spezialdiäten) ad libitum for 2 weeks.Drinking water was supplemented with 1% NaCl (w/v) with ad libitum access.Mice were habituated to handling during these 2 weeks.Afterward, samples were collected as described below.
IVF. IVF was performed at the Yale Genome Editing Center and the Transgenic Technologies Core Facility within the Forschungseinrichtungen für Experimentelle Medizin (FEM, Charité -Universitätsmedizin Berlin) using C57BL/6J females as egg donors and sperm from male Cacna1d I772M/+ mice.Sperm harvest and embryo transfer were performed as published (63).See Supplemental Methods for more information.
Behavioral phenotyping.All animals were habituated to handling by the experimenters for at least 2 weeks prior to behavior testing.For isradipine treatment, mice were randomly assigned to 2 different groups, and experimenters were blinded to the 2 groups but not genotype.In isradipine treatment, mice were given sweetened yogurt supplemented with 12.5 mg/kg isradipine once daily.Vascal uno (5 mg) capsules (CHEPLAPHARM Arzneimittel GmbH) containing a slow-release formulation of isradipine were opened, the powder was mixed with sweetened yogurt and weighed for dosing.In the control group, mice were given sweetened yogurt once daily.
The general health status of the mice was assessed by a SHIRPA test modified from (64).Phenotyping included the Rotarod test, open-field test (64), novel object recognition, 3-chamber test, nest construction test (64), buried food test (64), social proximity test, and HCS over 24 hours (64,65).We also performed mating behavior monitoring (66).The behavior was recorded with a video camera and assessed as described (67,68).See Supplemental Methods for more detailed information on the performed tests and their analysis.

Figure 1 .
Figure 1.Generation of Cacna1d Ile772Met/+ mice.(A) Topology of the Cacna1d gene (isoform ENSMUST00000238504.1) with Exons as blue boxes and intronic segments as thin lines.Introns longer than 2,000 bp are truncated.The sequence encoding the first 18 amino acids of exon 16 (encoding Ile772) is shown below.Substitution of ATG (Met; orange) for ATC (Ile; blue) was performed using CRISPR/Cas9 editing.The PAM site (gray background) as well as the gRNA (black box) used for mutagenesis are highlighted.(B) Sequencing of genomic DNA obtained from ear biopsies shows the heterozygous mutation.(C) Sequencing of cDNA from adrenal gland demonstrates expression of the mutant RNA.

Figure 5 .
Figure 5. Calcium imaging in acute adrenal slices to determine the response of the zona glomerulosa to potassium and angiotensin II.(A and B) Representative image of a WT (A) or Ile772Met/+ (B) adrenal slice stained with Fura-2 AM (top).On the bottom, representative traces are shown.Scale bars: 10 μm.(C) Mean calcium concentrations in zona glomerulosa cells determined by imaging of Fura-2 AM-stained slices.The extracellular concentration of angiotensin II was varied between 2 and 500 pM in the presence of either 3 (left) or 5 mM K + (right) (likelihood ratio test of linear mixed models; exact statistical values are given in Table 2; *P <0.05, **P < 0.01).

Figure 7 .
Figure 7. Cacna1d Ile772Met/+ mice show hyperlocomotion in response to changes in ambient light but are unable to maintain a hanging position at the top of the cage.(A-C) Course of 3 groups of home-cage behaviors over time for 24 hours (see Supplemental Figure 5 for all other groups).Cacna1d Ile772Met/+ mice show hyperlocomotion as indicated by longer distances traveled (A; likelihood ratio test of linear mixed models; n WT = 19, n Het = 17; P adj = 0.02, z = 0.085) together with increased periods spent moving around (B; likelihood ratio test of linear mixed models; n WT = 19, n Het = 17;P adj = 0.02, z = 0.00208).Hanging from the lid of the cage is significantly reduced in Cacna1d Ile772Met/+ mice compared with WT (C; likelihood ratio test of linear mixed models; n WT = 19, n Het = 17; P adj = 0.04, z = 2.631).For all statistical models, genotype was used as fixed and recording as well as time as random factors.All P values are adjusted for 10 comparisons (including the remaining behaviors shown in Supplemental Figure5) using the FDR method.Data are shown as mean ± 95% CI.