偵測細胞膜內外單價陽離子濃度

Abstract

生物利用不同的離子來維持各種生理活性。如鈣離子參與神經傳導物質的釋放以及肌肉收縮。鈉離子是細胞外液中濃度最高的離子，對於調節血壓和維持血液循環的平衡扮演重要的角色，血清中的鈉離子增加會使血壓升高;而鈉離子和鉀離子的共同合作使神經衝動維持正常的傳遞。鉀離子是細胞內含量最多的離子，負責調控細胞膜的通透性以及細胞生長等多種細胞活性；最重要的是，鉀離子決定靜止膜電位以調控神經細胞的興奮性。由於細胞在組織間彼此靠得很緊密，當微環境中離子濃度改變時，細胞間彼此會互相影響。我們感興趣的是了解在細胞膜兩側的鈉、鉀離子濃度變化並確認鈉鉀離子對於細胞間微環境的影響。我們首先利用鈉離子專一性的螢光染劑，研究細胞內鈉離子的濃度變化。研究結果顯示，細胞內的鈉離子基本濃度是16.5 ± 1.6 mM，而在10 μM 麩胺酸刺激神經細胞後，神經細胞內鈉離子濃度上升到154.0 ± 68.2 mM，再慢慢回到基礎值; 而細胞內鉀離子基本濃度是142.5 ± 43.5 mM，而在10 μM 麩胺酸刺激神經細胞後，神經細胞內鉀離子濃度下降到10.6 ± 0.8 mM，但卻不再上升。而為了研究細胞膜表面的鉀離子濃度，我們將可與鉀離子結合的適體(aptamer)修飾在矽奈米線場效電晶體上。數據顯示此適體對於不同的鹼金族離子有不同的親和力：鉀 (Kd = 7.9 ± 0.4 mM) > 銫 (Kd = 12.1 ± 0.5 mM) > 鈉 (Kd = 24.8 ± 4.1 mM) >> 鋰 (Kd = 189.0 ± 44.5 mM)。最近我們正試著將細胞置於矽奈米線上，以偵測細胞膜表面的鉀離子濃度。綜合以上測量結果，可以讓我們對於神經細胞間相互作用有更詳細的了解。

An organism utilizes different ions to support various physiological activities. Ca2+ participates in neurotransmitters release and muscle contraction. Na+ is the ion with highest concentration in the body fluid; the increase in the serum Na+ concentration elevates the blood pressure. In conjugation with K+, Na+ is involved in the transmission of nerve impulses. K+ is the most abundant ion inside the cell and regulates many cell activities like membrane permeability, growth, etc. Most importantly, K+ determines the resting membrane potential to modulate the neuron excitability. Because cells are in close contact with each other in tissues, the ion concentration changes at the interstitial microenvironment between cells will affect each other. Therefore, we are interested in understanding the concentrations of Na+ and K+ at both sides of the plasma membrane to verify their contributions to the microenvironment. We first used the Na+ specific fluorescence indicators to investigate the changes of intracellular Na+ concentration ([Na+]i) in primary-cultured neurons. The basal [Na+]i was 16.5 ± 1.6 mM and elevated to 154.0 ± 68.2 mM when stimulated by glutamate (10 μM); the concentration decline gradually afterwards. We used the K+ specific fluorescence indicators to investigate the changes of intracellular K+ concentration ([K+]i) in primary-cultured neurons. The basal [K+]i was 142.5 ± 43.5 mM and decreased to 10.6 ± 0.8 mM when stimulated by glutamate (10 μM); however, the [K+]i did not recover after the stimulation in 2 min. To investigate the K+ efflux at the membrane surface, we modified a K+-specific aptamer on the silicon nanowire field-effect transistor. The binding of K+ onto the apatamer changes the field effect surrounding the nanowire resulting in the conductivity changes. The data shows that the functionalized device responded to different alkali ions with various affinities: K+ (Kd = 7.9 ± 0.4 mM) > Cs+ (Kd = 12.1 ± 0.5 mM) > Na+ (Kd = 24.8 ± 4.1 mM) >> Li+ (Kd = 189.0 ± 44.5 mM). We are currently testing the K+ concentration at the membrane surface by anchoring cell onto the silicon nanowire. These measurements of the ion concentrations at the micro-environment will provide detail information in understanding the neuron-neuron interactions.