RESUMO
The S-specific pollen rejection response in Nicotiana depends on the interaction between S-RNase and a suite of SLF proteins. However, the biochemical pathway requires other essential proteins. One of them is the stigmatic protein NaStEP, which belongs to the Kunitz-type protease inhibitor family. Within the pollen tubes, NaStEP is a positive regulator of HT-B stability, likely inhibiting its degradation and, additionally, interacts with NaSIPP, a mitochondrial phosphate carrier. To gain a deeper understanding of the biochemical role of NaStEP in pollen rejection, we evaluated whether the activity of NaStEP as protease inhibitor is specific to a particular type of protease and whether it has the function of a voltage-dependent channel (VDC) blocker. Our findings indicate that, in vitro, NaStEP inhibits a subtilisin-like protease in an irreversible manner, but not other proteases, such as thermolysin and papain. Furthermore, we found that subtilisin processes the native NaStEP (24 kDa) into two lower molecular weight peptides of 21 and 14 kDa. Moreover, when we incubated NaStEP along with Xenopus leavis oocytes expressing the voltage-dependent potassium channel Kv 1.3, the current was blocked, indicating that NaStEP acts as a VDC blocker. These data allow us to propose NaStEP acts as a key molecule with two functions, one protecting HT-B from degradation by inhibiting a subtilisin-like protease and the second one by forming a complex with a mitochondrial VDC that could destabilize the mitochondria to trigger cell death, which would reinforce S-specific pollen rejection in Nicotiana.
Assuntos
Nicotiana , Proteínas de Plantas , Sequência de Aminoácidos , Moduladores de Transporte de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inibidores de Proteases , Nicotiana/genética , Nicotiana/metabolismoRESUMO
TASK channels belong to the family of K(+) channels with 4 transmembrane segments and 2 pore domains (4TM/2P) per subunit. These channels have been related to apoptosis in cerebellar granule neurons (CGN), as well as cancer in other tissues. TASK current is regulated by hormones, neurotransmitters, anesthetics and divalent cations, which are not selective. Recently, there has been found some organic compounds that inhibit TASK current selectively. In order to find other modulators, we report here a group of five dihydropyrrolo[2,1-a]isoquinolines (DPIs), four of them with putative anticancer activity, that were evaluated on TASK-1 and TASK-3 channels. The compounds 1, 2 and 3 showed IC50 < 320 µM on TASK-1 and TASK-3, intermediate activity on TASK-1/TASK-3 heterodimer, moderate effect over hslo and TREK-1 (500 µM), and practically not inhibition on Shaker-IR, herg and IRK2.1 potassium channels, when they were expressed heterologously in Xenopus laevis oocytes. In rat CGN, 500 µM of these three compounds induced a decrement by >39% of the TASK-carried leak current. Finally, only compound 1 showed significant protection (â¼36%) against apoptotic death of CGN induced by K(+) deprivation. These results suggest that DPI compounds could be potential candidates for designing new selective inhibitors of TASK channels.
Assuntos
Isoquinolinas/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Pirróis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Isoquinolinas/química , Camundongos , Estrutura Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Pirróis/química , Ratos , Ratos Wistar , Superfamília Shaker de Canais de Potássio/metabolismo , Xenopus laevisRESUMO
A novel peptide from Centruroides noxius Hoffmann scorpion venom was isolated and sequenced. The 37 amino acid peptide belongs to the charybdotoxin sub-family (alphaKTx1) and was numbered member 11. alphaKTx1.11 has 75% sequence identity with iberiotoxin and 54% with charybdotoxin. alphaKTx1.11 revealed specificity for mammalian MaxiK channels (hSlo), thus, was named slotoxin. Slotoxin blocks the MaxiK pore-forming alpha subunit reversibly (K(d)=1.5 nM). Slotoxin association with alpha+beta (beta1 or beta4) channels was approximately 10 times slower than iberiotoxin and charybdotoxin, leading to a lack of effect on alpha+beta4 when tested at 100 nM for 5 min. Thus, slotoxin is a better tool to distinguish MaxiK alpha+beta complexes.