RESUMO
The sequence of a novel cGMP-regulated, tetrameric, K(+) selective channel (Sp-tetraKCNG) was discovered in the sea urchin Strongylocentrotus purpuratus. The Sp-tetraKCNG is a single polypeptide made of four KCNG domains similar to voltage-dependent Na(+) and Ca(2+) channels. Each KCNG domain has six transmembrane segments (S1-S6), the ion pore having the K(+) selectivity signature GYGD and a cyclic nucleotide-binding domain (CNBD). This novel channel is evolutionary located between K(+)-selective and voltage-dependent EAG channels and voltage-independent cationic CNG channels. Bilayer reconstitutions demonstrate such a cGMP-regulated K(+) selective channel in sea urchin spermatozoa.
Assuntos
GMP Cíclico/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio/metabolismo , Espermatozoides/metabolismo , Strongylocentrotus purpuratus/fisiologia , Animais , Sítios de Ligação , Cátions/metabolismo , Bicamadas Lipídicas/metabolismo , Masculino , Dados de Sequência Molecular , Canais de Potássio/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Análise de Sequência de Proteína , Espermatozoides/química , Espermatozoides/citologiaRESUMO
Shaker, a voltage-dependent K+ channel, is enriched in the mushroom bodies (MBs), the locus of olfactory learning in Drosophila. Mutations in the shaker locus are known to alter excitability, neurotransmitter release, synaptic plasticity, and olfactory learning. However, a direct link of Shaker channels to MB intrinsic neuron (MBN) physiology has not been documented. We found that transcripts for shab, shaw, shaker, and shal, among which only Shaker and Shal have been reported to code for A-type currents, are present in the MBs. The electrophysiological data showed that the absence of functional Shaker channels modifies the distribution of half-inactivation voltages (V(i1/2)) in the MBNs, indicating a segregation of Shaker channels to only a subset (approximately 28%) of their somata. In harmony with this notion, we found that approximately one-fifth of MBNs lacking functional Shaker channels displayed dramatically slowed-down outward current inactivation times and reduced peak-current amplitudes. Furthermore, whereas all MBNs were sensitive to 4-aminopyridine, a nonspecific A-type current blocker, a subset of neurons (approximately 24%) displayed little sensitivity to a Shal-specific toxin. This subset of neurons displaying toxin-insensitive outward currents had more depolarized V(i1/2) values attributable to Shaker channels. Our findings provide the first direct evidence that altered Shaker channel function disrupts MBN physiology in Drosophila. To our surprise, the experimental data also indicate that Shaker channels segregate to a minor fraction of MB neuronal somata (20-30%), and that Shal channels contribute the somatic A-type current in the majority of MBNs.