Functional characterization of two novel germline mutations of the KCNJ5 gene in hypertensive patients without primary aldosteronism but with ACTH-dependent aldosterone hypersecretion.

BACKGROUND: Germline mutations of the KCNJ5 gene encoding Kir3·4, a member of the inwardly rectifying K+ channel, have been identified in 'normal' adrenal glands, patients with familial hyperaldosteronism (FH) type III, aldosterone-producing adenomas (APAs) and sporadic cases of primary aldosteronism (PA). OBJECTIVE: To present two novel KCNJ5 gene mutations in hypertensive patients without PA, but with Adrenocorticotropic hormone (ACTH)-dependent aldosterone hypersecretion. DESIGN AND PATIENTS: Two hypertensive patients without PA, who exhibited enhanced ACTH-dependent response of aldosterone secretion, underwent genetic testing for the presence of the CYP11B1/CYP11B2 chimeric gene and KCNJ5 gene mutations. Genomic DNA was isolated from peripheral white blood cells, and the exons of the entire coding regions of the above genes were amplified and sequenced. Electrophysiological studies were performed to determine the effect of identified mutation(s) on the membrane reversal potentials. Structural biology studies were also carried out. RESULTS: Two novel germline heterozygous KCNJ5 mutations, p.V259M and p.Y348N, were detected in the two subjects. Electrophysiological studies showed that the Y348N mutation resulted in significantly less negative reversal potentials, suggesting loss of ion selectivity, while the V259M mutation did not affect the Kir3.4 current. In the mutated structural biology model, the N348 mutant resulted in significant loss of the ability for hydrogen bonding, while the M259 mutant was capable of establishing weaker interactions. The CYP11B1/CYP11B2 chimeric gene was not detected. CONCLUSIONS: These findings expand on the clinical spectrum of phenotypes associated with KCNJ5 mutations and implicate these mutations in the pathogenesis of hypertension associated with increased aldosterone response to ACTH stimulation.

Matrix Metalloproteinase-9 Regulates Neuronal Circuit Development and Excitability.

In early postnatal development, naturally occurring cell death, dendritic outgrowth, and synaptogenesis sculpt neuronal ensembles into functional neuronal circuits. Here, we demonstrate that deletion of the extracellular proteinase matrix metalloproteinase-9 (MMP-9) affects each of these processes, resulting in maladapted neuronal circuitry. MMP-9 deletion increases the number of CA1 pyramidal neurons but decreases dendritic length and complexity. Parallel changes in neuronal morphology are observed in primary visual cortex and persist into adulthood. Individual CA1 neurons in MMP-9(-/-) mice have enhanced input resistance and a significant increase in the frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSCs). Additionally, deletion of MMP-9 significantly increases spontaneous neuronal activity in awake MMP-9(-/-) mice and enhances response to acute challenge by the excitotoxin kainate. Our data document a novel role for MMP-9-dependent proteolysis: the regulation of several aspects of circuit maturation to constrain excitability throughout life.

DPP6 domains responsible for its localization and function.

Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K(+) channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Here we show that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER.

Loss of signal transducer and activator of transcription 3 (STAT3) signaling during elevated activity causes vulnerability in hippocampal neurons.

Chronically altered levels of network activity lead to changes in the morphology and functions of neurons. However, little is known of how changes in neuronal activity alter the intracellular signaling pathways mediating neuronal survival. Here, we use primary cultures of rat hippocampal neurons to show that elevated neuronal activity impairs phosphorylation of the serine/threonine kinase, Erk1/2, and the activation of signal transducer and activator of transcription 3 (STAT3) by phosphorylation of serine 727. Chronically stimulated neurons go through apoptosis when they fail to activate another serine/threonine kinase, Akt. Gain- and loss-of-function experiments show that STAT3 plays the key role directly downstream from Erk1/2 as the alternative survival pathway. Elevated neuronal activity resulted in increased expression of a tumor suppressor, p53, and its target gene, Bax. These changes are observed in Kv4.2 knock-out mouse hippocampal neurons, which are also sensitive to the blockade of TrkB signaling, confirming that the alteration occurs in vivo. Thus, this study provides new insight into a mechanism by which chronic elevation of activity may cause neurodegeneration.

KCNJ5 mutations in the National Institutes of Health cohort of patients with primary hyperaldosteronism: an infrequent genetic cause of Conn's syndrome.

KCNJ5 mutations were recently described in primary hyperaldosteronism (PH or Conn's syndrome). The frequency of these mutations in PH and the way KCNJ5 defects cause disease remain unknown. A total of 53 patients with PH have been seen at the National Institutes of Health over the last 12 years. Their peripheral and tumor DNAs (the latter from 16 that were operated) were screened for KCNJ5 mutations; functional studies on the identified defects were performed after transient transfection. Only two mutations were identified, and both in the tumor DNA only. There were no germline sequencing defects in any of the patients except for known synonymous variants of the KCNJ5 gene. One mutation was the previously described c.G451C alteration; the other was a novel one in the same codon: c.G451A; both lead to the same amino acid substitution (G151R) in the KCNJ5 protein. Functional studies confirmed previous findings that both mutations caused loss of channel selectivity and a positive shift in the reversal potential. In conclusion, the KCNJ5 protein was strongly expressed in the zona glomerulosa of normal adrenal glands but showed variable expression in the aldosterone-producing adenomas with and without mutation. The rate of KCNJ5 mutations among patients with PH and/or their tumors is substantially lower than what was previously reported. The G151R amino acid substitution appears to be the most frequent one so far detected in PH, despite additional nucleotide changes. The mutation causes loss of this potassium channel's selectivity and may assist in the design of new therapies for PH.

Differential cycling rates of Kv4.2 channels in proximal and distal dendrites of hippocampal CA1 pyramidal neurons.

The heterogeneous expression of voltage-gated channels in dendrites suggests that neurons perform local microdomain computations at different regions. It has been shown that A-type K(+) channels have a nonuniform distribution along the primary apical dendrite in CA1 pyramidal neurons, increasing with distance from the soma. Kv4.2 channels, which are responsible for the somatodendritic A-type K(+) current in CA1 pyramidal neurons, shape local synaptic input, and regulate the back-propagation of APs into dendrites. Experiments were performed to test the hypothesis that Kv4.2 channels are differentially trafficked at different regions along the apical dendrite during basal activity and upon stimulation in CA1 neurons. Proximal (50-150 µm from the soma, primary and oblique) and distal (>200 µm) apical dendrites were selected. The fluorescence recovery after photobleaching (FRAP) technique was used to measure basal cycling rates of EGFP-tagged Kv4.2 (Kv4.2g). We found that the cycling rate of Kv4.2 channels was one order of magnitude slower at both primary and oblique dendrites between 50 and 150 µm from the soma. Kv4.2 channel cycling increased significantly at 200 to 250 µm from the soma. Expression of a Kv4.2 mutant lacking a phosphorylation site for protein kinase-A (Kv4.2gS552A) abolished this distance-dependent change in channel cycling; demonstrating that phosphorylation by PKA underlies the increased mobility in distal dendrites. Neuronal stimulation by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) treatment increased cycling of Kv4.2 channels significantly at distal sites only. This activity-dependent increase in Kv4.2 cycling at distal dendrites was blocked by expression of Kv4.2gS552A. These results indicate that distance-dependent Kv4.2 mobility is regulated by activity-dependent phosphorylation of Kv4.2 by PKA.

A kinase anchor protein 150 (AKAP150)-associated protein kinase A limits dendritic spine density.

The A kinase anchor protein AKAP150 recruits the cAMP-dependent protein kinase (PKA) to dendritic spines. Here we show that in AKAP150 (AKAP5) knock-out (KO) mice frequency of miniature excitatory post-synaptic currents (mEPSC) and inhibitory post-synaptic currents (mIPSC) are elevated at 2 weeks and, more modestly, 4 weeks of age in the hippocampal CA1 area versus litter mate WT mice. Linear spine density and ratio of AMPAR to NMDAR EPSC amplitudes were also increased. Amplitude and decay time of mEPSCs, decay time of mIPSCs, and spine size were unaltered. Mice in which the PKA anchoring C-terminal 36 residues of AKAP150 are deleted (D36) showed similar changes. Furthermore, whereas acute stimulation of PKA (2-4 h) increases spine density, prolonged PKA stimulation (48 h) reduces spine density in apical dendrites of CA1 pyramidal neurons in organotypic slice cultures. The data from the AKAP150 mutant mice show that AKAP150-anchored PKA chronically limits the number of spines with functional AMPARs at 2-4 weeks of age. However, synaptic transmission and spine density was normal at 8 weeks in KO and D36 mice. Thus AKAP150-independent mechanisms correct the aberrantly high number of active spines in juvenile AKAP150 KO and D36 mice during development.

AKAP79/150 impacts intrinsic excitability of hippocampal neurons through phospho-regulation of A-type K+ channel trafficking.

Kv4.2, as the primary α-subunit of rapidly inactivating, A-type voltage-gated K(+) (Kv) channels expressed in hippocampal CA1 pyramidal dendrites, plays a critical role in regulating their excitability. Activity-dependent trafficking of Kv4.2 relies on C-terminal protein kinase A (PKA) phosphorylation. A-kinase-anchoring proteins (AKAPs) target PKA to glutamate receptor and ion channel complexes to allow for discrete, local signaling. As part of a previous study, we showed that AKAP79/150 interacts with Kv4.2 complexes and that the two proteins colocalize in hippocampal neurons. However, the nature and functional consequence of their interaction has not been previously explored. Here, we report that the C-terminal domain of Kv4.2 interacts with an internal region of AKAP79/150 that overlaps with its MAGUK (membrane-associated guanylate kinase)-binding domain. We show that AKAP79/150-anchored PKA activity controls Kv4.2 surface expression in heterologous cells and hippocampal neurons. Consistent with these findings, disrupting PKA anchoring led to a decrease in neuronal excitability, while preventing dephosphorylation by the phosphatase calcineurin resulted in increased excitability. These results demonstrate that AKAP79/150 provides a platform for dynamic PKA regulation of Kv4.2 expression, fundamentally impacting CA1 excitability.

KChIP4a regulates Kv4.2 channel trafficking through PKA phosphorylation.

Voltage-gated potassium (Kv) channels play important roles in regulating the excitability of myocytes and neurons. Kv4.2 is the primary alpha-subunit of the channel that produces the A-type K(+) current in CA1 pyramidal neurons of the hippocampus, which is critically involved in the regulation of dendritic excitability and plasticity. K(+) channel-interacting proteins, KChIPs (KChIP1-4), associate with the N-terminal of Kv4.2 and modulate the channel's biophysical properties, turnover rate and surface expression. In the present study, we investigated the role of Kv4.2 C-terminal PKA phosphorylation site S552 in the KChIP4a-mediated effects on Kv4.2 channel trafficking. We found that while interaction between Kv4.2 and KChIP4a does not require PKA phosphorylation of Kv4.2(S552), phosphorylation of this site is necessary for both enhanced stabilization and membrane expression of Kv4.2 channel complexes produced by KChIP4a. Enhanced surface expression and protein stability conferred by co-expression of Kv4.2 with other KChIP isoforms did not require PKA phosphorylation of Kv4.2 S552. Finally, we identify A-kinase anchoring proteins (AKAPs) as Kv4.2 binding partners, allowing for discrete local PKA signaling. These data demonstrate that PKA phosphorylation of Kv4.2 plays an important role in the trafficking of Kv4.2 through its specific interaction with KChIP4a.

Protein kinase a mediates activity-dependent Kv4.2 channel trafficking.

The A-type potassium channel subunit Kv4.2 influences hippocampal function through regulation of dendritic excitability, and changes in Kv4.2 surface expression alter synaptic plasticity. Recent data from our laboratory demonstrate that EGFP (enhanced green fluorescent protein)-tagged Kv4.2 channels located in dendritic spines are internalized in an activity-dependent manner after synaptic stimulation and during chemically induced long-term potentiation. However, the molecular trigger for Kv4.2 internalization remains unknown. Here we examined the role of protein kinase A (PKA) in Kv4.2 activity-dependent trafficking. In hippocampal neurons, PKA activation with forskolin or 8-Br-cAMP induced Kv4.2 internalization from dendritic spines, whereas PKA inhibition with H89 prevented AMPA-induced internalization. Furthermore, introduction of a point mutation at the C-terminal PKA phosphorylation site of Kv4.2 (S552A) prevented the AMPA-induced internalization of Kv4.2. Together, these data demonstrate that Kv4.2 activity-dependent internalization requires PKA phosphorylation of Kv4.2 at serine 522.