Abstract-The excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K ϩ ) conductances. Although studies suggest that background K ϩ currents carried by 2-pore domain K ϩ channels are important regulators of resting membrane potential in PASMC, their role in human PASMC is unknown. Our study tested the hypothesis that TASK-1 leak K ϩ channels contribute to the K ϩ current and resting membrane potential in human PASMC. We used the whole-cell patch-clamp technique and TASK-1 small interfering RNA (siRNA). Noninactivating K ϩ current performed by TASK-1 K ϩ channels were identified by current characteristics and inhibition by anandamide and acidosis (pH 6.3), each resulting in significant membrane depolarization. Moreover, we showed that TASK-1 is blocked by moderate hypoxia and activated by treprostinil at clinically relevant concentrations. This is mediated via protein kinase A (PKA)-dependent phosphorylation of TASK-1. To further confirm the role of TASK-1 channels in regulation of resting membrane potential, we knocked down TASK-1 expression using TASK-1 siRNA. The knockdown of TASK-1 was reflected by a significant depolarization of resting membrane potential. Treatment of human PASMC with TASK-1 siRNA resulted in loss of sensitivity to anandamide, acidosis, alkalosis, hypoxia, and treprostinil. These results suggest that (1) TASK-1 is expressed in human PASMC; (2) TASK-1 is hypoxia-sensitive and controls the resting membrane potential, thus implicating an important role for TASK-1 K ϩ channels in the regulation of pulmonary vascular tone; and (3) treprostinil activates TASK-1 at clinically relevant concentrations via PKA, which might represent an important mechanism underlying the vasorelaxing properties of prostanoids and their beneficial effect in vivo. Key Words: pulmonary circulation Ⅲ potassium channels Ⅲ TASK-1 Ⅲ treprostinil Ⅲ hypoxic pulmonary vasoconstriction T he membrane potential of pulmonary artery smooth muscle cells (PASMC) is an important regulator of arterial tone. These cells have a resting membrane potential of approximately Ϫ65 to Ϫ50 mV in vitro, close to the predicted equilibrium potential for potassium (K ϩ ) ions. The opening of K ϩ channels in the PASMC membrane increases K ϩ efflux, which causes membrane hyperpolarization. This closes voltage-dependent Ca 2ϩ channels, decreasing Ca 2ϩ entry and leading to vasodilatation. Conversely, inhibition of K ϩ channels causes membrane depolarization, Ca 2ϩ entry, cell contraction, and vasoconstriction.Background or leak K ϩ -selective channels, as defined by a lack of time and voltage dependency, play an essential role in setting the resting membrane potential and input resistance in excitable cells. Two-pore domain K ϩ (2-PK) channels have been shown to conduct several leak K ϩ currents. The activity of 2-PK channels is strongly regulated by protons, protein kinases, and hypoxia. Alteration of K ϩ conductance can influence cellular activity via membrane potential changes.Both RT-PCR and Northern blot analyses p...

Spinal muscular atrophies (SMA, also known as hereditary motor neuropathies) and hereditary motor and sensory neuropathies (HMSN) are clinically and genetically heterogeneous disorders of the peripheral nervous system. Here we report that mutations in the TRPV4 gene cause congenital © 2009 Nature America, Inc. All rights reserved.Correspondence should be addressed to M.A.-G. (michaela.auergrumbach@medunigraz.at).. METHODS: Methods and any associated references are available in the online version of the paper at http://www.nature.com/ naturegenetics/. Accession codes. GenBank: human TRPV4 cDNA, NM_021625; human TRPV4, NP_067638 IsoA. Pfam: ankyrin repeat, PF00023.Note: Supplementary information is available on the Nature Genetics website. AUTHOR CONTRIBUTIONS: M.A.-G., S.U., J.S., M.E.M., A.H.C., K.J.D., C.M.A.v.R.-A., N.E.A., H.L., B.S.-W., R.P., C.L., G.W.P., H.J.S., H.K. and T.R.P. recruited the study participants, acquired clinical data, conducted neurological and neurophysiological evaluations and performed linkage analysis. M.A.-G, C.G., L.P. and C.F. carried out the Affymetrix array linkage studies and identified the mutations. A.O., Z.B. and B.T. designed, carried out and analyzed the electrophysiological and Ca 2+ -imaging studies. E.F. conducted immunofluorescence and immunohistochemistry studies. H.S. conducted fluorescence-activated cell sorting (FACS) and biotinylation studies. A.K. performed structural biology and biocomputing analyses. A.H.C., M.E.M. and H.K. participated in the data analysis and reviewed the manuscript. M.A.-G. and C.G. analyzed the data, designed and supervised the study and wrote the manuscript. Supplementary Fig. 1) and observed linkage to three chromosomal regions with log 10 of odds (lod) scores >2 for several SNP markers, including the chromosome 12q23-24 region (data not shown). We constructed haplotypes by including additional distantly related family members (right branch of the pedigree; Supplementary Fig. 1). The genetic interval transmitted with the disease resides between SNPs rs2374688 and rs35426 (Chr. 12: 106,197,054,429 bp; Supplementary Table 1) and overlaps with the intervals reported for risk of congenital distal SMA, SPSMA and HMSN2C 2-4 . Europe PMC Funders GroupIn an affected individual from family FAM_1, we began sequencing all protein-coding exons and exon-intron boundaries of 19 genes but initially observed only known SNPs (Supplementary Table 2). However, sequencing of all protein-coding exons of TRPV4 (transient receptor potential vanilloid 4; chr. 12: 108,705,277-108,755,595; reverse strand) revealed a heterozygous C-to-T nucleotide change at position 943 in exon 6 (Supplementary Fig. 2a), which is predicted to cause the substitution of arginine with tryptophan at position 315 of TRPV4 (R315W). We then screened DNA samples from additional families showing one of the phenotypes described above, including two families previously reported 1,3,4 . All affected individuals from the chromosome 12q23-24-linked family (here called FAM_2) described by...

The potassium channel TWIK-related acid sensitive potassium (TASK)-1 channel, together with other potassium channels, controls the low resting tone of pulmonary arteries. The Src family tyrosine kinase (SrcTK) may control potassium channel function in human pulmonary artery smooth muscle cells (hPASMCs) in response to changes in oxygen tension and the clinical use of a SrcTK inhibitor has resulted in partly reversible pulmonary hypertension.This study aimed to determine the role of SrcTK in hypoxia-induced inhibition of potassium channels in hPASMCs.We show that SrcTK is co-localised with the TASK-1 channel. Inhibition of SrcTK decreases potassium current density and results in considerable depolarisation, while activation of SrcTK increases potassium current in patch-clamp recordings. Moderate hypoxia and the SrcTK inhibitor decrease the tyrosine phosphorylation state of the TASK-1 channel. Hypoxia also decreases the level of phospho-SrcTK (tyr419) and reduces the co-localisation of the TASK-1 channel and phospho-SrcTK. Corresponding to this, hypoxia reduces TASK-1 currents before but not after SrcTK inhibition and, in the isolated perfused mouse lung, SrcTK inhibitors increase pulmonary arterial pressure.We propose that the SrcTK is a crucial factor controlling potassium channels, acting as a cofactor for setting a negative resting membrane potential in hPASMCs and a low resting pulmonary vascular tone.