At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa-like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na(+)/K(+) ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.

BackgroundCav1.3 voltage-gated L-type calcium channels (LTCCs) are part of postsynaptic neuronal signaling networks. They play a key role in brain function, including fear memory and emotional and drug-taking behaviors. A whole-exome sequencing study identified a de novo mutation, p.A749G, in Cav1.3 α1-subunits (CACNA1D), the second main LTCC in the brain, as 1 of 62 high risk–conferring mutations in a cohort of patients with autism and intellectual disability. We screened all published genetic information available from whole-exome sequencing studies and identified a second de novo CACNA1D mutation, p.G407R. Both mutations are present only in the probands and not in their unaffected parents or siblings.MethodsWe functionally expressed both mutations in tsA-201 cells to study their functional consequences using whole-cell patch-clamp.ResultsThe mutations p.A749G and p.G407R caused dramatic changes in channel gating by shifting (~15 mV) the voltage dependence for steady-state activation and inactivation to more negative voltages (p.A749G) or by pronounced slowing of current inactivation during depolarizing stimuli (p.G407R). In both cases, these changes are compatible with a gain-of-function phenotype.ConclusionsOur data, together with the discovery that Cav1.3 gain-of-function causes primary aldosteronism with seizures, neurologic abnormalities, and intellectual disability, suggest that Cav1.3 gain-of-function mutations confer a major part of the risk for autism in the two probands and may even cause the disease. Our findings have immediate clinical relevance because blockers of LTCCs are available for therapeutic attempts in affected individuals. Patients should also be explored for other symptoms likely resulting from Cav1.3 hyperactivity, in particular, primary aldosteronism.

L-type calcium channels (Cav1) represent one of the three major classes (Cav1–3) of voltage-gated calcium channels. They were identified as the target of clinically used calcium channel blockers (CCBs; so-called calcium antagonists) and were the first class accessible to biochemical characterization. Four of the 10 known α1 subunits (Cav1.1–Cav1.4) form the pore of L-type calcium channels (LTCCs) and contain the high-affinity drug-binding sites for dihydropyridines and other chemical classes of organic CCBs. In essentially all electrically excitable cells one or more of these LTCC isoforms is expressed, and therefore it is not surprising that many body functions including muscle, brain, endocrine, and sensory function depend on proper LTCC activity. Gene knockouts and inherited human diseases have allowed detailed insight into the physiological and pathophysiological role of these channels. Genome-wide association studies and analysis of human genomes are currently providing even more hints that even small changes of channel expression or activity may be associated with disease, such as psychiatric disease or cardiac arrhythmias. Therefore, it is important to understand the structure–function relationship of LTCC isoforms, their differential contribution to physiological function, as well as their fine-tuning by modulatory cellular processes.

The Ca(2+) channel alpha(1S) subunit (Ca(V)1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca(2+) release from internal stores and conducts a slowly activating Ca(2+) current. However, this Ca(2+) current is not essential for skeletal muscle EC coupling. Here, we identified a Ca(V)1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged alpha(1S) subunit and expressed it in dysgenic (alpha(1S)-null) myotubes. GFP-alpha(1S)Delta 29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca(2+) currents through GFP-alpha(1S)Delta 29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca(2+) influx contributed substantially to the depolarization-induced Ca(2+) transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary alpha(2)delta-1 subunit. Thus, characterizing the Ca(V)1.1 Delta 29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca(2+) channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.

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Auxiliary channel subunits regulate membrane expression and modulate current properties of voltageactivated Ca 2؉ channels and thus are involved in numerous important cell functions, including muscle contraction. Whereas the importance of the ␣ 1S , ␤ 1a , and ␥ Ca 2؉ channel subunits in skeletal muscle has been determined by using null-mutant mice, the role of the ␣ 2 ␦-1 subunit in skeletal muscle is still elusive. We addressed this question by small interfering RNA silencing of ␣ 2 ␦-1 in reconstituted dysgenic (␣ 1S -null) myotubes and in BC3H1 skeletal muscle cells. Immunofluorescence labeling of the ␣ 1S and ␣ 2 ␦-1 subunits and whole cell patch clamp recordings demonstrated that triad targeting and functional expression of the skeletal muscle Ca 2؉ channel were not compromised by the depletion of the ␣ 2 ␦-1 subunit. The amplitudes and voltage dependences of L-type Ca 2؉ currents and of the depolarization-induced Ca 2؉ transients were identical in control and in ␣ 2 ␦-1-depleted muscle cells. However, ␣ 2 ␦-1 depletion significantly accelerated the current kinetics, most likely by the conversion of slowly activating into fast activating Ca 2؉ channels. Reverse transcription-PCR analysis indicated that ␣ 2 ␦-1 is the exclusive isoform expressed in differentiated BC3H1 cells and that depletion of ␣ 2 ␦-1 was not compensated by the up-regulation of any other ␣ 2 ␦ isoform. Thus, in skeletal muscle the Ca 2؉ channel ␣ 2 ␦-1 subunit functions as a major determinant of the characteristic slow L-type Ca 2؉ current kinetics. However, this subunit is not essential for targeting of Ca 2؉ channels or for their primary physiological role in activating skeletal muscle excitation-contraction coupling.Voltage-activated Ca 2ϩ channels are important signaling proteins in many cellular processes including muscle contraction, secretion, synaptic function, and transcriptional regulation. Ca 2ϩ channels are composed of a pore-forming ␣ 1 subunit and the auxiliary ␣ 2 ␦, ␤, and ␥ subunits (1). Whereas the ␣ 1 subunits are responsible for voltage sensing and ion conduction, the auxiliary subunits have been implicated in functions of membrane targeting and modulation of channel properties (for review see Ref.2). Much of our current knowledge about the specific properties of Ca 2ϩ channel subunits has been obtained from heterologous expression in Xenopus oocytes and in mammalian expression systems. Moreover, null-mutant mice have provided important information about the roles of Ca 2ϩ channel subunits in native tissues.In skeletal muscle the voltage-activated Ca 2ϩ channel functions as a voltage sensor in excitation-contraction (EC) 1 coupling. The role of the slowly activating L-type Ca 2ϩ current, which is not necessary for the activation of skeletal muscle contraction, is not clear. A null-mutant of the skeletal muscle ␣ 1S subunit, the dysgenic mouse, lacks EC coupling and L-type Ca 2ϩ currents and dies at birth from respiratory failure (3). A knock-out mouse of the skeletal ␤ 1a Ca 2ϩ channel subunit results in a very simila...

Voltage-gated calcium channels (CaV) regulate numerous vital functions in nerve and muscle cells. To fulfill their diverse functions, the multiple members of the CaV channel family are activated over a wide range of voltages. Voltage sensing in potassium and sodium channels involves the sequential transition of positively charged amino acids across a ring of residues comprising the charge transfer center. In CaV channels, the precise molecular mechanism underlying voltage sensing remains elusive. Here we combined Rosetta structural modeling with site-directed mutagenesis to identify the molecular mechanism responsible for the specific gating properties of two CaV1.1 splice variants. Our data reveal previously unnoticed interactions of S4 arginines with an aspartate (D1196) outside the charge transfer center of the fourth voltage-sensing domain that are regulated by alternative splicing of the S3-S4 linker. These interactions facilitate the final transition into the activated state and critically determine the voltage sensitivity and current amplitude of these CaV channels.

In neurons L-type calcium currents function in gene regulation and synaptic plasticity, while excessive calcium influx leads to excitotoxicity and neurodegeneration. The major neuronal Ca V 1.2 L-type channels are localized in clusters in dendritic shafts and spines. Whereas Ca V 1.2 clusters remain stable during NMDA-induced synaptic depression, L-type calcium currents are rapidly downregulated during strong excitatory stimulation. Here we used fluorescence recovery after photobleaching (FRAP), live cell-labeling protocols, and single particle tracking (SPT) to analyze the turnover and surface traffic of Ca V 1.2 in dendrites of mature cultured mouse and rat hippocampal neurons, respectively. FRAP analysis of channels extracellularly tagged with superecliptic pHluorin (Ca V 1.2-SEP) demonstrated ϳ20% recovery within 2 min without reappearance of clusters. Pulse-chase labeling showed that membrane-expressed Ca V 1.2-HA is not internalized within1 h, while blocking dynamin-dependent endocytosis resulted in increased cluster density after 30 min. Together, these results suggest a turnover rate of clustered Ca V 1.2s on the hour time scale. Direct recording of the lateral movement in the membrane using SPT demonstrated that dendritic Ca V 1.2s show highly confined mobility with diffusion coefficients of ϳ0.005 m 2 s Ϫ1 . Consistent with the mobile Ca V 1.2 fraction observed in FRAP, a ϳ30% subpopulation of channels reversibly exchanged between confined and diffusive states. Remarkably, high potassium depolarization did not alter the recovery rates in FRAP or the diffusion coefficients in SPT analyses. Thus, an equilibrium of clustered and dynamic Ca V 1.2s maintains stable calcium channel complexes involved in activitydependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations.