RESUMO
Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.
Assuntos
Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Estruturas da Membrana Celular/metabolismo , Integrinas/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sódio/metabolismo , Animais , Células CHO , Proteínas de Transporte de Cátions/genética , Permeabilidade da Membrana Celular/fisiologia , Estruturas da Membrana Celular/genética , Cricetinae , Cricetulus , Humanos , Integrinas/genética , Transporte de Íons/fisiologia , Transdução de Sinais/fisiologia , Trocador de Sódio e Cálcio/genética , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
The cyclic calcium release and uptake during calcium oscillation are thought to result from calcium-induced calcium release (CICR); however, it is unclear, especially in nonexcitable cells, how the initial calcium mobilization that triggers CICR occurs. We report here a novel mechanism, other than conventional calcium channels or the phopholipase C-inositol trisphosphate system, for initiating calcium oscillation downstream of integrin signaling. Upon integrin alphaIIbbeta3 binding to fibrinogen ligand or the disintegrin rhodostomin, sodium-proton exchanger NHE1 and sodium-calcium exchanger NCX1 are actively transported to the plasma membrane, and they become physically coupled to integrin alphaIIbbeta3. Lipid raft-dependent mechanisms modulate the membrane targeting and formation of the NHE1-integrin alphaIIbbeta3-NCX1 protein complex. NHE1 and NCX1 within such protein complex are functionally coupled, such that a local increase of sodium concentration caused by NHE1 can drive NCX1 to generate sodium efflux in exchange for calcium influx. The resulting calcium increase inside the cell can then trigger CICR as a prelude to calcium oscillation downstream of integrin alphaIIbbeta3 signaling. Fluorescence resonance energy transfer based on fluorescence lifetime measurements is employed here to monitor the intermolecular interactions among NHE1-integrin alphaIIbbeta3-NCX1, which could not be properly detected using conventional biochemical assays.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Microdomínios da Membrana/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Relógios Biológicos/fisiologia , Células CHO , Proteínas de Transporte de Cátions/genética , Cricetinae , Cricetulus , Fibrinogênio/genética , Fibrinogênio/metabolismo , Humanos , Inositol 1,4,5-Trifosfato , Microdomínios da Membrana/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Transporte Proteico/fisiologia , Sódio/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
BACKGROUND: In our previous study, an A-type voltage-gated K(+) channel, K(v)3.4, was found more frequently expressed in oral squamous cell carcinoma (OSCC) when compared with non-cancerous matched oral tissue. An OSCC cell line, OECM-1, was found to have moderate level of K(v)3.4 expression. METHODS: To further elucidate the roles of K(v)3.4 for the involvement of neoplastic process, we amplified K(v)3.4 coding sequence by reverse transcriptase polymerase chain reaction (RT-PCR), constructed an expression vector carrying this sequence and then stably transfected into OECM-1 OSCC cells. RESULTS: We demonstrated the integration and constitutive expression of K(v)3.4 in the cell. A unique A-type current elicited by such expression in OECM-1 cells was defined by patch clamp analysis. This current pattern can be reversibly blocked by an A-type K(+) channel blocker 2 mM 4-aminopyridine (4-AP). The acquisition of K(v)3.4 activity in OECM-1 cells bestowed growth advantage. However, in 3T3 cell, transfected K(v)3.4 caused only limited increase of growth without forming transformation foci. CONCLUSION: The present study established a stable keratinocyte system carrying functional K(v)3.4 and increase of growth, by which the anti-K(v)3.4 modalities for potential OSCC control can be further investigated.
