Somatic and germline CACNA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism

Abstract

Adrenal aldosterone-producing adenomas (APAs) constitutively produce the salt-retaining hormone aldosterone and are a common cause of severe hypertension. Recurrent mutations in the potassium channel gene KCNJ5 that result in cell depolarization and Ca(2+) influx cause ∼40% of these tumors. We identified 5 somatic mutations (4 altering Gly403 and 1 altering Ile770) in CACNA1D, encoding a voltage-gated calcium channel, among 43 APAs without mutated KCNJ5. The altered residues lie in the S6 segments that line the channel pore. Both alterations result in channel activation at less depolarized potentials; Gly403 alterations also impair channel inactivation. These effects are inferred to cause increased Ca(2+) influx, which is a sufficient stimulus for aldosterone production and cell proliferation in adrenal glomerulosa. We also identified de novo germline mutations at identical positions in two children with a previously undescribed syndrome featuring primary aldosteronism and neuromuscular abnormalities. These findings implicate gain-of-function Ca(2+) channel mutations in APAs and primary aldosteronism.

Figure 1

CACNA1D mutations in aldosterone-producing adenomas and primary aldosteronism. (a) Sequences of tumor and blood genomic DNA, and (where available) tumor cDNA, of CACNA1D codons 402–404 in APA37, APA31, APA65 and APA59, and of codons 769–771 in APA29. Mutations are present in tumor only, and expressed in cDNA. Sequencing the products of cloned PCR products confirmed the presence of identified mutations in APAs 31, 65 and 59. (b) Pedigrees of kindreds with germline CACNA1D mutations. Affected individuals are shown as filled symbols. The corresponding Sanger sequences are depicted to the right. (c) Conservation of Gly403 and Ile770 in orthologs and paralogs. S6, S6 segment; ‘h’, high-voltage activated; ‘l’, low-voltage activated. Residues conserved among all homologs are marked in yellow, and positions conserved in ≥90% of all homologs in both repeats are marked in green. Residues associated with known gain of function mutations in human diseases^–, are marked in purple.

Figure 2

Transmembrane structure of Ca_V1.3. CACNA1D encodes the pore-forming α₁ subunit of a voltage-gated calcium channel. These channels feature four homologous repeats (I–IV) with 6 transmembrane segments (S1-S6) and a membrane-associated loop between segments S5 and S6. The five APA and two germline CACNA1D mutations identified in this study are located at the end of S6 segments implicated in channel gating.

Figure 3

Immunohistochemistry of Ca_V1.3 in human adrenal gland. Human adrenal gland was stained with anti-Ca_V1.3 (Sigma) (a,c) or anti-Dab2 (b,d, an adrenal glomerulosa marker), and sections were counterstained with haematoxylin. Ca_V1.3 is expressed in adrenal glomerulosa. (a,b), scale bar represents 500 μm; (c,d), 100 μm. C, capsule; G, glomerulosa; F, fasciculata; R, reticularis.

Figure 4

Ca_V1.3 mutations shift the voltage-dependence of activation to more hyperpolarized potentials. (a) Representative whole cell recordings of HEK293T cells transiently expressing WT or mutant Ca_V1.3 together with β_2a and α₂δ₁ subunits (vertical bar: 50 pA). Currents were elicited by voltage pulses between −60 mV and +20 mV including the peak current amplitude (arrow). (b) Voltage dependence of normalized peak current amplitudes (I_max) of WT (n = 7), Gly403Asp (n = 5), Gly403Arg (n = 7) or Ile770Met (n = 6) channels. The voltage dependence of activation is shifted to more hyperpolarized potentials in Gly403Asp, Gly403Arg and Ile770Met channels. (c) Activation (filled symbols) and inactivation (open symbols) curves of WT and Ile770Met channels. Activation and inactivation of Ile770Met channels are both shifted to more hyperpolarized potentials. Activation curves were calculated from b, while the inactivation curves were calculated from fits of the inactivation time courses. Data are presented as mean ± s.e.m.