Hyperpolarization-activated calcium channels at the tip of Arabidopsis root hairs

The root hair elongative growth phase (“tip growth”), like that of other tip-growing systems such as pollen tubes, algal rhizoids, and fungal hyphae, is associated with an apex-high cytosolic free calcium ([Ca(2+)](c)) gradient generated by a local Ca(2+) influx at the tip. This gradient has been shown to be a fundamental regulator of tip growth. Here, we have performed patch-clamp experiments at root hair apices of Arabidopsis thaliana (after localized cell wall laser ablation) to characterize the plasma membrane Ca(2+) channels implicated in the tip Ca(2+) influx. We have identified a hyperpolarization-activated Ca(2+) conductance. This conductance is selective for Ca(2+) over K(+) and Cl(−) (P(Ca)/P(K) = 15; P(Ca)/P(Cl) = 25) and is fully blocked by <100-μM trivalent cations (La(3+), Al(3+), Gd(3+)). The selectivity sequence among divalent cations (determined by comparisons of the channel unitary conductance) is Ba(2+) > Ca(2+) (22 pS in 10 mM) ≈ Mg(2+) > Mn(2+). This conductance was operative at typical growing hair apical resting membrane potentials. Moreover, it was seen to be down-regulated in growing hair subapical regions, as well as at the tip of mature hairs (known not to exhibit Ca(2+) influx). We therefore propose that this inward-rectifying Ca(2+) conductance is inherently involved in the apical Ca(2+) influx of growing hairs. The observed enhancement of the conductance by increased [Ca(2+)](c) may form part of a positive feedback system for continued apical Ca(2+) influx during tip growth.

根毛伸长生长阶段（又称"顶端生长"）与其他顶端生长系统（如花粉管、藻类假根及真菌菌丝）类似，均依赖于由顶端局部钙离子内流所建立的胞质游离钙（[Ca²⁺](c)）顶端梯度。该梯度已被证实是顶端生长的核心调控因子。本研究通过对拟南芥根毛顶端（经局部细胞壁激光消融处理后）开展膜片钳实验，对参与顶端钙离子内流的质膜钙离子通道进行功能表征。本研究鉴定出一种超极化激活的钙离子电导。该电导对钙离子相较于钾离子与氯离子具有选择通透性（P(Ca)/P(K)=15；P(Ca)/P(Cl)=25），且可被浓度低于100μM的三价阳离子（La³+、Al³+、Gd³+）完全阻断。通过对比单通道电导所得的二价阳离子选择通透性序列为：Ba²+ > Ca²+（10mM条件下为22pS）≈ Mg²+ > Mn²+。该电导在生长中根毛顶端的典型静息膜电位下即可发挥功能。此外，该电导在生长中根毛的亚顶端区域以及成熟根毛的顶端（已知不发生钙离子内流）中均被下调。因此我们提出，该内向整流钙离子电导天然参与生长中根毛的顶端钙离子内流过程。实验观察到的胞质游离钙浓度升高对该电导的增强效应，可能构成顶端生长过程中维持顶端钙离子内流的正反馈系统的一部分。