RESUMEN
A cDNA encoding a potassium channel of the two-pore domain family (K(2P), KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK(2P) cannot be placed into any of the established functional groups of mammalian K(2P) channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK(2P). In whole cells, non-inactivating, voltage-independent, outwardly rectifying K(+) currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC(50) â¼30 µmol l(-1)), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK(2P) but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K(2P) channels, the sponge K(2P) channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pK(a) 8.18) activated the AquK(2P) channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K(2P) channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K(2P) channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA.
Asunto(s)
Álcalis/farmacología , Organismos Acuáticos/fisiología , Ácidos Grasos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Poríferos/fisiología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Secuencia de Aminoácidos , Animales , Organismos Acuáticos/efectos de los fármacos , Ácido Araquidónico/farmacología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Datos de Secuencia Molecular , Ósmosis/efectos de los fármacos , Filogenia , Poríferos/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/química , Homología de Secuencia de Aminoácido , Temperatura , Xenopus laevisRESUMEN
Ion channels establish and regulate membrane potentials in excitable and non-excitable cells. How functional diversification of ion channels contributed to the evolution of nervous systems may be understood by studying organisms at key positions in the evolution of animal multicellularity. We have carried out the first analysis of ion channels cloned from a marine sponge, Amphimedon queenslandica. Phylogenetic comparison of sequences encoding for poriferan inward-rectifier K(+) (Kir) channels suggests that Kir channels from sponges, cnidarians and triploblastic metazoans each arose from a single channel and that duplications arose independently in the different groups. In Xenopus oocytes, AmqKirA and AmqKirB produced K(+) currents with strong inward rectification, as seen in the mammalian Kir2 channels, which are found in excitable cells. The pore properties of AmqKir channels demonstrated strong K(+) selectivity and block by Cs(+) and Ba(2+). We present an original analysis of sponge ion channel physiology and an examination of the phylogenetic relationships of this channel with other cloned Kir channels.