Abstract
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.