DeFazio R A, Keros S, Quick M W, Hablitz J J
Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Neurosci. 2000 Nov 1;20(21):8069-76. doi: 10.1523/JNEUROSCI.20-21-08069.2000.
Chloride (Cl(-)) homeostasis is critical for many cell functions including cell signaling and volume regulation. The action of GABA at GABA(A) receptors is primarily determined by the concentration of intracellular Cl(-). Developmental regulation of intracellular Cl(-) results in a depolarizing response to GABA in immature neocortical neurons and a hyperpolarizing or shunting response in mature neocortical neurons. One protein that participates in Cl(-) homeostasis is the neuron-specific K(+)-Cl(-) cotransporter (KCC2). Thermodynamic considerations predict that in the physiological ranges of intracellular Cl(-) and extracellular K(+) concentrations, KCC2 can act to either extrude or accumulate Cl(-). To test this hypothesis, we examined KCC2 function in pyramidal cells from rat neocortical slices in mature (18-28 d postnatal) and immature (3-6 d postnatal) rats. Intracellular Cl(-) concentration was estimated from the reversal potential of whole-cell currents evoked by local application of exogenous GABA. Both increasing and decreasing the extracellular K(+) concentration resulted in a concomitant change in intracellular Cl(-) concentration in neurons from mature rats. KCC2 inhibition by furosemide caused a change in the intracellular Cl(-) concentration that depended on the concentration of pipette Cl(-); in recordings with low pipette Cl(-), furosemide lowered intracellular Cl(-), whereas in recordings with elevated pipette Cl(-), furosemide raised intracellular Cl(-). In neurons from neonatal rats, manipulation of extracellular K(+) had no effect on intracellular Cl(-) concentration, consistent with the minimal KCC2 mRNA levels observed in neocortical neurons from immature animals. These data demonstrate a physiologically relevant and developmentally regulated role for KCC2 in Cl(-) homeostasis via both Cl(-) extrusion and accumulation.
氯离子（Cl(-)）稳态对于包括细胞信号传导和体积调节在内的许多细胞功能至关重要。γ-氨基丁酸（GABA）在GABA(A)受体上的作用主要由细胞内Cl(-)的浓度决定。细胞内Cl(-)的发育调节导致未成熟新皮层神经元对GABA产生去极化反应，而在成熟新皮层神经元中则产生超极化或分流反应。一种参与Cl(-)稳态的蛋白质是神经元特异性钾氯共转运体（KCC2）。热力学考虑预测，在细胞内Cl(-)和细胞外K(+)浓度的生理范围内，KCC2可以起到排出或积累Cl(-)的作用。为了验证这一假设，我们研究了成熟（出生后18 - 28天）和未成熟（出生后3 - 6天）大鼠新皮层切片中锥体细胞的KCC2功能。通过局部应用外源性GABA诱发的全细胞电流的反转电位来估计细胞内Cl(-)浓度。细胞外K(+)浓度的增加和降低都会导致成熟大鼠神经元细胞内Cl(-)浓度的相应变化。呋塞米对KCC2的抑制作用导致细胞内Cl(-)浓度的变化，这取决于移液管中Cl(-)的浓度；在移液管中Cl(-)浓度较低的记录中，呋塞米降低了细胞内Cl(-)浓度，而在移液管中Cl(-)浓度升高的记录中，呋塞米提高了细胞内Cl(-)浓度。在新生大鼠的神经元中，细胞外K(+)的操作对细胞内Cl(-)浓度没有影响，这与未成熟动物新皮层神经元中观察到的KCC2 mRNA水平极低一致。这些数据表明，KCC2通过Cl(-)的排出和积累在Cl(-)稳态中发挥生理相关且受发育调节的作用。
