Staruschenko Alexandr V, Vedernikova Elena A
Institute of Cytology, Russian Academy of Sciences, 194064, St Petersburg, Russia.
J Physiol. 2002 May 15;541(Pt 1):81-90. doi: 10.1113/jphysiol.2001.015222.
Cell-attached and inside-out patch-clamp methods were employed to identify and characterize mechanosensitive (MS) ionic channels in the plasma membrane of human myeloid leukaemia K562 cells. A reversible activation of gadolinium-blockable mechanogated currents in response to negative pressure application was found in 58 % of stable patches (n = 317). I-V relationships measured with a sodium-containing pipette solution showed slight inward rectification. Data analysis revealed the presence of two different populations of channels that were distinguishable by their conductance properties (17.2 +/- 0.3 pS and 24.5 +/- 0.5 pS), but were indistinguishable with regard to their selective and pharmacological properties. Ion-substitution experiments indicated that MS channels in leukaemia cells were permeable to cations but not to anions and do not discriminate between Na(+) and K(+). The channels were fully impermeable to large organic cations such as Tris(+) and N-methyl-D-glucamine ions (NMDG(+)). Ca(2+) permeation and blockade of MS channels were examined using pipettes containing different concentrations of Ca(2+). In the presence of 2 mM CaCl(2), when other cations were impermeant, both outward and inward single-channel currents were observed; the I-V relationship showed a unitary conductance of 7.7 +/- 1.0 pS. The relative permeability value, P(Ca)/P(K), was equal to 0.75, as estimated at physiological Ca(2+) concentrations. Partial or full inhibition of inward Ca(2+) currents through MS channels was observed at higher concentrations of external Ca(2+) (10 or 20 mM). No MS channels were activated when using a pipette containing 90 mM CaCl(2). Monovalent mechanogated currents were not significantly affected by extracellular Ca(2+) at concentrations within the physiological range (0-2 mM), and at some higher Ca(2+) concentrations.
采用细胞贴附式和外向膜片钳技术,对人髓系白血病K562细胞膜上的机械敏感(MS)离子通道进行鉴定和特性分析。在58%的稳定膜片(n = 317)中发现,负压作用可引起钆可阻断的机械门控电流可逆激活。用含钠的电极内液测得的电流-电压关系显示出轻微的内向整流。数据分析表明,存在两种不同类型的通道,可通过其电导特性(17.2±0.3 pS和24.5±0.5 pS)区分,但在选择性和药理学特性方面无法区分。离子置换实验表明,白血病细胞中的MS通道对阳离子通透,对阴离子不通透,且对Na(+)和K(+)无选择性。该通道对诸如Tris(+)和N-甲基-D-葡糖胺离子(NMDG(+))等大的有机阳离子完全不通透。使用含不同浓度Ca(2+)的电极,研究了Ca(2+)对MS通道的通透和阻断作用。在存在2 mM CaCl(2)且其他阳离子不通透的情况下,观察到外向和内向单通道电流;电流-电压关系显示单位电导为7.7±1.0 pS。在生理Ca(2+)浓度下估计,相对通透率值P(Ca)/P(K)等于0.75。在较高的细胞外Ca(2+)浓度(10或20 mM)下,观察到通过MS通道的内向Ca(2+)电流部分或完全被抑制。当使用含90 mM CaCl(2)的电极时,未激活MS通道。在生理范围内(0 - 2 mM)以及一些较高Ca(2+)浓度下,细胞外Ca(2+)对单价机械门控电流无显著影响。