We have isolated from the rat cerebellum cDNA library a complementary DNA encoding a new member of the tandem pore K ؉ channel family. Its amino acid sequence shares 54% identity with that of TASK-1, but less than 30% with those of TASK-2 and other tandem pore K ؉ channels (TWIK, TREK, TRAAK). Therefore, the new clone was named TASK-3. Reverse transcriptase-polymerase chain reaction analysis showed that TASK-3 mRNA is expressed in many rat tissues including brain, kidney, liver, lung, colon, stomach, spleen, testis, and skeletal muscle, and at very low levels in the heart and small intestine. When expressed in COS-7 cells, TASK-3 exhibited a time-independent, noninactivating K ؉ -selective current. Single-channel conductance was 27 pS at ؊60 mV and 17 pS at 60 mV in symmetrical 140 mM KCl. TASK-3 current was highly sensitive to changes in extracellular pH (pH o ), a hallmark of the TASK family of K ؉ channels. Thus, a change in pH o from 7.2 to 6.4 and 6.0 decreased TASK-3 current by 74 and 96%, respectively. Mutation of histidine at position 98 to aspartate abolished pH o sensitivity. TASK-3 was blocked by barium (57%, 3 mM), quinidine (37%, 100 M), and lidocaine (62%, 1 mM). Thus, TASK-3 is a new member of the acidsensing K ؉ channel subfamily (TASK).Potassium (K ϩ ) channels are involved in a variety of cellular functions including regulation of neuronal firing rate, heart rate, muscle contraction, and hormone secretion. Mammalian K ϩ channels can now be grouped into three main structural classes with each subunit possessing two, four, or six transmembrane segments (1-3). A structurally different K ϩ channel having eight transmembrane segments has been cloned from yeast (4 -6), but a similar channel subunit has not been identified in the mammalian system. Despite the structural diversity, all K ϩ channel subunits share a conserved P domain that is essential for providing K ϩ selectivity (7-9). In Caenorhabditis elegans, ϳ50 putative K ϩ channels subunits possessing two pore-forming domains and four transmembrane segments (2P/ 4TM) 1 have been identified by searching the genome sequences (10, 11). Recent cloning efforts have led to the identification of several members of the 2P/4TM K ϩ channels. Open rectifier K ϩ channel (ORK1) from Drosophila melanogaster and tandem of P domains in a weak inward rectifying K ϩ channel (TWIK-1) from human kidney were the first two members of this family to be cloned (12, 13). Recent studies now indicate that TWIK-1 does not form a functional ion channel, whereas the open rectifier K ϩ channel 1 does (14, 15). Subsequently, other members of this family were cloned using expressed sequence tags identified by searching the GenBank TM data base for TWIK-1-like sequences or using degenerate primers designed to amplify a DNA fragment with sequences homologous to TWIK-1. Electrophysiological studies of 4TM K ϩ channels suggest that most behave as background K ϩ currents (ORK1, TASK, TREK, TRAAK), although some have additional properties such as sensitivity to mechanical stretch, fr...

Recently, several mammalian K ؉ channel subunits (TWIK, TREK-1, TRAAK, and TASK) possessing four transmembrane segments and two pore-forming domains have been identified. We report the cloning of a new member of this tandem-pore K ؉ channel from a rat cerebellum cDNA library. It is a 538-amino acid protein and shares 65% amino acid sequence identity with TREK-1. Therefore, the new clone was named TREK-2. Unlike TREK-1, whose mRNA has been reported to be expressed in many different tissues, TREK-2 mRNA is expressed mainly in the cerebellum, spleen, and testis as judged by reverse transcriptase-polymerase chain reaction and Northern blot analysis. Expression of TREK-2 in COS-7 cells induced a time-independent and non-inactivating K ؉ -selective current. TREK-2 was partially blocked (36%) by 2 mM Ba 2؉. In symmetrical 150 mM KCl, the single-channel conductances were 110 picosiemens at ؊40 mV and 68 picosiemens at ؉40 mV, and the mean open time was 0.9 ms at ؊40 mV. TREK-2 was activated by membrane stretch or acidic pH. At ؊40 mm Hg pressure, channel activity increased 10-fold above the basal level. TREK-2 was also activated by arachidonic acid and other naturally occurring unsaturated free fatty acids. These results show that TREK-2 is a new member of the tandem-pore K ؉ channel family and belongs to the class of mechanosensitive and fatty acid-stimulated K ؉ channels.

A mammalian K(+) channel subunit (TBAK-1/TASK-1) containing two pore domains and four transmembrane segments and whose mRNA is highly expressed in the heart has been cloned recently. TBAK-1 and TASK-1 are identical except for the additional nine amino acids in the NH(2) terminus of TBAK-1. We examined their kinetic properties, pH sensitivity, and regional cardiac mRNA expression and determined whether a native cardiac K(+) channel with similar kinetic properties was present. When TBAK-1 or TASK-1 was transiently expressed in COS-7 cells, time- and voltage-independent whole cell currents were observed. Single-channel conductances of TBAK-1 and TASK-1 were 14.6 +/- 1.0 and 13.8 +/- 2.8 pS, respectively, at -80 mV in 140 mM extracellular K(+), and the mean open times were 0.8 +/- 0.1 and 0.6 +/- 0.1 ms, respectively. Both TBAK-1 and TASK-1 were highly sensitive to extracellular pH such that a decrease from 7.2 to 6.4 reduced their open probability (P(o)) by 81 +/- 14% and 80 +/- 16%, whereas a decrease in intracellular pH from 7.2 to 6.4 reduced the P(o) by 42 +/- 10% and 47 +/- 12%, respectively. TBAK-1/TASK-1 mRNA was expressed in all regions of the rat heart, with the highest level of expression in the right atrium. A 14-pS K(+) channel with kinetic properties similar to those of TBAK-1/TASK-1 was identified in rat atrial and ventricular cells. These results indicate that TBAK-1/TASK-1 represents a functional native K(+) channel in the rat heart.

TRAAK is a member of the tandem-pore K+ channel family, and is expressed mainly in the brain. Using rat TRAAK (rTRAAK), we studied its single-channel kinetics, interactive modes of activation, and the role of the C-terminus on its pressure-, fatty-acid- and pH-sensitivity. When expressed in COS-7 cells, rTRAAK showed a mildly inwardly rectifying single-channel current/voltage relationship in symmetrical 140 mM KCl. Unlike TREK-1 and TREK-2, which are activated by acidic conditions, rTRAAK was activated by alkali conditions, such that a change in intracellular pH from 7.3 to 8.3 and 8.8 increased channel activity 5- and 14-fold, respectively. Pressure and alkali produced a strong synergistic activation, and pressure and arachidonic acid (AA) produced a mild synergistic activation. The only additive effect was observed with alkali and AA. Replacing the C-terminus of rTRAAK with that of TASK-1 or TASK-3 did not affect the response to pressure, AA or alkali. In contrast, replacing the C-terminus of TREK-2 with that of TASK-3 abolished the sensitivity to AA and acid, but not to pressure. These results show that rTRAAK is an alkali-sensing K+ channel that shows synergistic activation with pressure, and that the mechanism of activation of rTRAAK and TREK by free fatty acids are different.

PurposeBoth telomere length and mitochondrial function are accepted as reflective indices of aging. Recent studies have shown that telomere dysfunction may influence impaired mitochondrial biogenesis and function. However, there has been no study regarding the possible association between telomere and mitochondrial function in humans. Therefore, the purpose of the study was to identify any relationships between mitochondrial and telomere function.MethodsThe present study included 129 community-dwelling, elderly women. The leukocyte mitochondrial DNA copy number and telomere length were measured using a quantitative real-time polymerase chain reaction method. Anthropometric measurement, biochemical blood testing, a depression screening questionnaire using a 15-question geriatric depression scale (GDS-15), and a cognitive function test using the Korean version of the mini mental state examination (K-MMSE) were performed.ResultsLeukocyte mtDNA copy number was positively associated with telomere length (r=0.39, p=<0.0001) and K-MMSE score (r=0.06, p=0.02). Additionally, leukocyte mtDNA copy number was negatively correlated with GDS-15 score (r=-0.17, p=0.04). Age (r=-0.15, p=0.09), waist circumference (r=-0.16, p=0.07), and serum ferritin level (r=-0.13, p=0.07) tended to be inversely correlated with leukocyte mtDNA copy number. With a stepwise multiple regression analysis, telomere length was found to be an independent factor associated with leukocyte mtDNA copy number after adjustment for confounding variables including age, body mass index, waist circumference, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, hs-CRP, serum ferritin, HOMA-IR, K-MMSE, GDS-15, hypertension, diabetes, dyslipidemia, currently smoking, alcohol drinking, and regular exercise.ConclusionsThis study showed that leukocyte mtDNA copy number was positively correlated with leukocyte telomere length in community-dwelling elderly women. Our findings suggest that telomere function may influence mitochondrial function in humans.

BackgroundIon channels play a critical role in a wide variety of biological processes, including the development of human cancer. However, the overall impact of ion channels on tumorigenicity in breast cancer remains controversial.MethodsWe conduct microarray meta-analysis on 280 ion channel genes. We identify candidate ion channels that are implicated in breast cancer based on gene expression profiling. We test the relationship between the expression of ion channel genes and p53 mutation status, ER status, and histological tumor grade in the discovery cohort. A molecular signature consisting of ion channel genes (IC30) is identified by Spearman’s rank correlation test conducted between tumor grade and gene expression. A risk scoring system is developed based on IC30. We test the prognostic power of IC30 in the discovery and seven validation cohorts by both Cox proportional hazard regression and log-rank test.Results22, 24, and 30 ion channel genes are found to be differentially expressed with a change in p53 mutation status, ER status, and tumor histological grade in the discovery cohort. We assign the 30 tumor grade associated ion channel genes as the IC30 gene signature. We find that IC30 risk score predicts clinical outcome (P < 0.05) in the discovery cohort and 6 out of 7 validation cohorts. Multivariate and univariate tests conducted in two validation cohorts indicate that IC30 is a robust prognostic biomarker, which is independent of standard clinical and pathological prognostic factors including patient age, lymph node status, tumor size, tumor grade, estrogen and progesterone receptor status, and p53 mutation status.ConclusionsWe identified a molecular gene signature IC30, which represents a promising diagnostic and prognostic biomarker in breast cancer. Our results indicate that information regarding the expression of ion channels in tumor pathology could provide new targets for therapy in human cancers.

TREK-2, a member of the tandem-pore K+ channel family, is activated by membrane stretch, unsaturated free fatty acids and acidic conditions, and exhibits unique open channel kinetics. To identify the regions responsible for these properties, we studied the role of the cytoplasmic regions of TREK-2. Deletion of the N-terminus had no effect on any aspect of TREK-2 function. Deletion of the C-terminus or its substitution with that of TASK-3 abolished the sensitivity to free fatty acids and intracellular pH (pHi), and reduced the sensitivity to pressure. The regions that allow activation by free fatty acids and low pHi were localized to 25- and 10-amino-acid domains, respectively, close to the fourth transmembrane segment. Substitution of KKTKEE, a charged region near the proximal C-terminus, with uncharged amino acids produced little change in TREK-2 function; however, its deletion abolished sensitivity to fatty acids and low pH, indicating that this region is structurally very important. The TREK-2 C-terminus was also found to be critical for its channel opening in bursts as well as for its increased basal activity. Thus, the C-terminus endows TREK-2 with unique channel kinetics and the ability to be gated by free fatty acids and low pHi, and with increased mechanosensitivity.

Habitual physical exercise is associated with greater telomere length in postmenopausal women. This finding suggests that habitual physical exercise in postmenopausal women may reduce telomere attrition.