RESUMO
Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca2+ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP3) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca2+ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca2+ channels that facilitate a measurable Ca2+ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.
Assuntos
Compostos de Boro/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Molécula 1 de Interação Estromal/genética , Moléculas de Interação Estromal/genética , Animais , Compostos de Boro/síntese química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteína ORAI1/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Molécula 1 de Interação Estromal/antagonistas & inibidores , Moléculas de Interação Estromal/antagonistas & inibidoresRESUMO
Electrical pacemaker activity generates phasic contractions and motility patterns such as segmentation and peristalsis in the gastrointestinal tract. Pacemaker currents are generated in interstitial cells of Cajal (ICC), which release Ca2+ from intracellular stores that stimulates Ca2+-activated Cl- channels (CaCCs) in the plasma membrane. Thus, Ca2+ stores must be maintained to sustain pacemaker activity. Store-operated Ca2+ entry (SOCE) facilitates the refilling of Ca2+ stores by a mechanism dependent upon interactions between STIM and Orai proteins. We investigated the role of SOCE in ICC pacemaker activity. Reintroduction of extracellular Ca2+ in store-depleted ICC resulted in CaCC activation. Blocking CaCCs revealed an inwardly rectifying current with properties of a Ca2+ release-activated current (ICRAC). An inhibitory peptide that interfered with the STIM-Orai interaction blocked ICRAC in HEK 293 cells expressing STIM1 and Orai1 and blocked spontaneous transient inward currents (STICs) and slow wave currents in ICC. STICs, which are fundamental pacemaker events in ICC, were blocked by an Orai antagonist. Imaging of Ca2+ transients linked to pacemaker activity in ICC in intact muscles showed that the Orai antagonist blocked Ca2+ transients in ICC. These data suggest that Ca2+ recovery through STIM-Orai interactions is necessary to maintain ICC pacemaker activity.