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.

A low concentration of ethanol impairs learning but not motor and sensory behavior in Drosophila larvae.

Drosophila melanogaster has proven to be a useful model system for the genetic analysis of ethanol-associated behaviors. However, past studies have focused on the response of the adult fly to large, and often sedating, doses of ethanol. The pharmacological effects of low and moderate quantities of ethanol have remained understudied. In this study, we tested the acute effects of low doses of ethanol (∼7 mM internal concentration) on Drosophila larvae. While ethanol did not affect locomotion or the response to an odorant, we observed that ethanol impaired associative olfactory learning when the heat shock unconditioned stimulus (US) intensity was low but not when the heat shock US intensity was high. We determined that the reduction in learning at low US intensity was not a result of ethanol anesthesia since ethanol-treated larvae responded to the heat shock in the same manner as untreated animals. Instead, low doses of ethanol likely impair the neuronal plasticity that underlies olfactory associative learning. This impairment in learning was reversible indicating that exposure to low doses of ethanol does not leave any long lasting behavioral or physiological effects.

A novel point mutation in the KCNJ5 gene causing primary hyperaldosteronism and early-onset autosomal dominant hypertension.

CONTEXT: Aldosterone production in the adrenal zona glomerulosa is mainly regulated by angiotensin II, [K(+)], and ACTH. Genetic deletion of subunits of K(+)-selective leak (KCNK) channels TWIK-related acid sensitive K(+)-1 and/or TWIK-related acid sensitive K(+)-3 in mice results in primary hyperaldosteronism, whereas mutations in the KCNJ5 (potassium inwardly rectifying channel, subfamily J, member 5) gene are implicated in primary hyperaldosteronism and, in certain cases, in autonomous glomerulosa cell proliferation in humans. OBJECTIVE: The objective of the study was to investigate the role of KCNK3, KCNK5, KCNK9, and KCNJ5 genes in a family with primary hyperaldosteronism and early-onset hypertension. PATIENTS AND METHODS: Two patients, a mother and a daughter, presented with severe primary hyperaldosteronism, bilateral massive adrenal hyperplasia, and early-onset hypertension refractory to medical treatment. Genomic DNA was isolated 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. RESULTS: Sequencing of the KCNJ5 gene revealed a single, heterozygous guanine to thymine (G → T) substitution at nucleotide position 470 (n.G470T), resulting in isoleucine (I) to serine (S) substitution at amino acid 157 (p.I157S). This mutation results in loss of ion selectivity, cell membrane depolarization, increased Ca(2+) entry in adrenal glomerulosa cells, and increased aldosterone synthesis. Sequencing of the KCNK3, KCNK5, and KCNK9 genes revealed no mutations in our patients. CONCLUSIONS: These findings explain the pathogenesis in a subset of patients with severe hypertension and implicate loss of K(+) channel selectivity in constitutive aldosterone production.

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.

Olfactory conditioning in the third instar larvae of Drosophila melanogaster using heat shock reinforcement.

Adult Drosophila melanogaster has long been a popular model for learning and memory studies. Now the larval stage of the fruit fly is also being used in an increasing number of classical conditioning studies. In this study, we employed heat shock as a novel negative reinforcement for larvae and obtained high learning scores following just one training trial. We demonstrated heat-shock conditioning in both reciprocal and non-reciprocal paradigms and observed that the time window of association for the odor and heat shock reinforcement is on the order of a few minutes. This is slightly wider than the time window for electroshock conditioning reported in previous studies, possibly due to lingering effects of the high temperature. To test the utility of this simplified assay for the identification of new mutations that disrupt learning, we examined flies carrying mutations in the dnc gene. While the sensitivity to heat shock, as tested by writhing, was similar for wild type and dnc homozygotes, dnc mutations strongly diminished learning. We confirmed that the learning defect in dnc flies was indeed due to mutation in the dnc gene using non-complementation analysis. Given that heat shock has not been employed as a reinforcement for larvae in the past, we explored learning as a function of heat shock intensity and found that optimal learning occurred around 41 °C, with higher and lower temperatures both resulting in lower learning scores. In summary, we have developed a very simple, robust paradigm of learning in fruit fly larvae using heat shock reinforcement.