scholarly journals CACNA1HM1549V Mutant Calcium Channel Causes Autonomous Aldosterone Production in HAC15 Cells and Is Inhibited by Mibefradil

Endocrinology ◽  
2016 ◽  
Vol 157 (8) ◽  
pp. 3016-3022 ◽  
Author(s):  
Esther N. Reimer ◽  
Gudrun Walenda ◽  
Eric Seidel ◽  
Ute I. Scholl
Keyword(s):  
Calcium Channel ◽  
Primary Aldosteronism ◽  
Cancer Cell Line ◽  
Fold Increase ◽  
Extracellular Potassium ◽  
Channel Blockers ◽  
Adrenocortical Cancer ◽  
Transfected Cells ◽  
Channel Inactivation ◽  
Aldosterone Production

We recently demonstrated that a recurrent gain-of-function mutation in a T-type calcium channel, CACNA1HM1549V, causes a novel Mendelian disorder featuring early-onset primary aldosteronism and hypertension. This variant was found independently in five families. CACNA1HM1549V leads to impaired channel inactivation and activation at more hyperpolarized potentials, inferred to cause increased calcium entry. We here aimed to study the effect of this variant on aldosterone production. We heterologously expressed empty vector, CACNA1HWT and CACNA1HM1549V in the aldosterone-producing adrenocortical cancer cell line H295R and its subclone HAC15. Transfection rates, expression levels, and subcellular distribution of the channel were similar between CACNA1HWT and CACNA1HM1549V. We measured aldosterone production by an ELISA and CYP11B2 (aldosterone synthase) expression by real-time PCR. In unstimulated cells, transfection of CACNA1HWT led to a 2-fold increase in aldosterone levels compared with vector-transfected cells. Expression of CACNA1HM1549V caused a 7-fold increase in aldosterone levels. Treatment with angiotensin II or increased extracellular potassium levels further stimulated aldosterone production in both CACNA1HWT- and CACNA1HM1549V-transfected cells. Similar results were obtained for CYP11B2 expression. Inhibition of CACNA1H channels with the T-type calcium channel blocker Mibefradil completely abrogated the effects of CACNA1HWT and CACNA1HM1549V on CYP11B2 expression. These results directly link CACNA1HM1549V to increased aldosterone production. They suggest that calcium channel blockers may be beneficial in the treatment of a subset of patients with primary aldosteronism. Such blockers could target CACNA1H or both CACNA1H and the L-type calcium channel CACNA1D that is also expressed in the adrenal gland and mutated in patients with primary aldosteronism.

scholarly journals Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Ute I Scholl ◽  
Gabriel Stölting ◽  
Carol Nelson-Williams ◽  
Alfred A Vichot ◽  
Murim Choi ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Primary Aldosteronism ◽  
Early Onset ◽  
De Novo ◽  
Incomplete Penetrance ◽  
De Novo Mutations ◽  
Channel Inactivation ◽  
Aldosterone Production ◽  
Genome Level ◽  
Insight Into

Many Mendelian traits are likely unrecognized owing to absence of traditional segregation patterns in families due to causation by de novo mutations, incomplete penetrance, and/or variable expressivity. Genome-level sequencing can overcome these complications. Extreme childhood phenotypes are promising candidates for new Mendelian traits. One example is early onset hypertension, a rare form of a global cause of morbidity and mortality. We performed exome sequencing of 40 unrelated subjects with hypertension due to primary aldosteronism by age 10. Five subjects (12.5%) shared the identical, previously unidentified, heterozygous CACNA1HM1549V mutation. Two mutations were demonstrated to be de novo events, and all mutations occurred independently. CACNA1H encodes a voltage-gated calcium channel (CaV3.2) expressed in adrenal glomerulosa. CACNA1HM1549V showed drastically impaired channel inactivation and activation at more hyperpolarized potentials, producing increased intracellular Ca2+, the signal for aldosterone production. This mutation explains disease pathogenesis and provides new insight into mechanisms mediating aldosterone production and hypertension.

Characterization of a mutated KCNJ5 gene, G387R, in unilateral primary aldosteronism

2021 ◽  
Vol 67 (4) ◽  
pp. 203-215
Author(s):  
Jeff S Chueh ◽  
Kang-Yung Peng ◽  
Vin-Cent Wu ◽  
Shuo-Meng Wang ◽  
Chieh-Kai Chan ◽  
...  
Keyword(s):  
Primary Aldosteronism ◽  
Bioinformatics Analysis ◽  
Genomic Analysis ◽  
Ion Current ◽  
Mutant Channel ◽  
Transfected Cells ◽  
Aldosterone Production ◽  
Mutant Cells ◽  
Electrophysiological Experiment

Somatic mutation in the KCNJ5 gene is a common driver of autonomous aldosterone overproduction in aldosterone-producing adenomas (APA). KCNJ5 mutations contribute to a loss of potassium selectivity, and an inward Na+ current could be detected in cells transfected with mutated KCNJ5. Among 223 unilateral primary aldosteronism (uPA) individuals with a KCNJ5 mutation, we identified 6 adenomas with a KCNJ5 p.Gly387Arg (G387R) mutation, previously unreported in uPA patients. The six uPA patients harboring mutant KCNJ5-G387R were older, had a longer hypertensive history, and had milder elevated preoperative plasma aldosterone levels than those APA patients with more frequently detected KCNJ5 mutations. CYP11B2 immunohistochemical staining was only positive in three adenomas, while the other three had co-existing multiple aldosterone-producing micronodules. The bioinformatics analysis predicted that function of the KCNJ5-G387R mutant channel could be pathological. However, the electrophysiological experiment demonstrated that transfected G387R mutant cells did not have an aberrantly stimulated ion current, with lower CYP11B2 synthesis and aldosterone production, when compared to that of the more frequently detected mutant KCNJ5-L168R transfected cells. In conclusion, mutant KCNJ5-G387R is not a functional KCNJ5 mutation in unilateral PA. Compared with other KCNJ5 mutations, the observed mildly elevated aldosterone expression actually hindered the clinical identification of clinical unilateral PA. The KCNJ5-G387R mutation needs to be distinguished from functional KCNJ5 mutations during genomic analysis in APA evaluation because of its functional silence.

Calcium channel involvement in GABAB receptor-mediated inhibition of GABA release in area CA1 of the rat hippocampus

1995 ◽  
Vol 74 (1) ◽  
pp. 43-53 ◽  
Author(s):  
V. A. Doze ◽  
G. A. Cohen ◽  
D. V. Madison
Keyword(s):  
Calcium Channels ◽  
Action Potential ◽  
Calcium Channel ◽  
Calcium Channel Blockers ◽  
Gabab Receptor ◽  
Gaba Release ◽  
Extracellular Potassium ◽  
Channel Blockers ◽  
Voltage Dependent ◽  
Subtype Selective

1. Experiments were performed in rat hippocampal slices to examine the nature of GABAergic inhibition of inhibitory synaptic transmission. In these experiments the effects of the gamma-aminobutyric acid-B (GABAB) receptor agonist, baclofen, and of subtype-selective calcium channel blockers were tested with the use of intracellular recordings of evoked inhibitory postsynaptic potentials (IPSPs) and whole cell recordings of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs). 2. Baclofen inhibited evoked and spontaneous (action-potential-dependent) monosynaptic GABAA-mediated IPSPs and IPSCs but had no effect on the frequency of tetrodotoxin-resistant (action-potential-independent) miniature IPSCs recorded in CA1 pyramidal neurons. 3. Depolarizing GABAergic synaptic terminals by raising the extracellular potassium concentration caused an increase in action-potential-independent miniature IPSC frequency that could be inhibited by either baclofen or cadmium, a blocker of voltage-dependent calcium channels. In addition, under these depolarizing conditions, cadmium occluded the baclofen inhibition of miniature IPSCs. These data suggest that baclofen reduces only depolarization-induced, not quantal, GABA release and that it does so by decreasing presynaptic voltage-dependent calcium influx. 4. Experiments with subtype-selective calcium channel blockers demonstrate that the presynaptic action of baclofen was mediated through both omega-conotoxin-GVIA-sensitive and omega-agatoxin-IVA-sensitive, but not dihydropyridine-sensitive calcium channels.

Spike aftercurrents in R15 of Aplysia: their relationship to slow inward current and calcium influx

1984 ◽  
Vol 51 (2) ◽  
pp. 387-403 ◽  
Author(s):  
D. V. Lewis
Keyword(s):  
Calcium Channel ◽  
Intracellular Calcium ◽  
Calcium Channel Blockers ◽  
Calcium Influx ◽  
Extracellular Calcium ◽  
Slow Inward Current ◽  
Extracellular Potassium ◽  
Channel Blockers ◽  
Tetraacetic Acid ◽  
Inward Current

Spikes in the bursting neuron, R15, are followed by depolarizing afterpotentials (35) and often by delayed hyperpolarizing afterpotentials as well. Placing the cell in a voltage clamp after a spike allows measurement of the depolarizing aftercurrent (DAC) and hyperpolarizing aftercurrent (HAC) that underlie the afterpotentials. Subthreshold depolarizations give rise to small DACs and HACs. The DAC and the slow inward current (SIC) of R15 are reduced or blocked in a similar manner by many experimental manipulations, e.g., application of dopamine, zero-calcium seawater, zero-sodium seawater, or calcium-channel blockers (Mn2+ and La3+), or cooling the cell from 21-22 degrees C to 10 degrees C. Neither the DAC nor the SIC were blocked by tetrodotoxin (100 uM) and neither was sensitive to altered extracellular potassium. Both the DAC and SIC become larger as the holding potential of the cell is progressively depolarized from -70 to -40 mV. DACs are sensitive to the injection of intracellular calcium chelators (EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N1-tetraacetic acid) or EDTA [ethylenedinitrilo)tetraacetic acid]. DAC amplitude is approximately 90% reduced by intracellular EGTA concentration near 1mM. In contrast, the SIC is unchanged or much less affected by the calcium buffers. DACs are also more sensitive to low (1 mM) extracellular calcium than is the SIC. The HAC is also a calcium-dependent current. It is blocked by any experimental manipulation reducing calcium influx or intracellular calcium accumulation, i.e., reduced extracellular calcium, calcium-channel blockers, or intracellular EGTA. We suggest that the DAC and the SIC are carried by the same conductance mechanism. In the case of the DAC, the conductance might be activated by a rise in intracellular calcium activity accompanying the spike and, in the case of the SIC, depolarization per se may be the most important activating condition.

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