Ca2+ Influx into Lily Pollen Grains Through a Hyperpolarization-activated Ca2+-permeable Channel Which Can be Regulated by Extracellular CaM

Abstract

Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca²⁺ influx in maintaining the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and the features of the Ca²⁺ influx pathway in germinating pollen grains of Lilium davidii D. [Ca²⁺]_c decreased when Ca²⁺ influx was inhibited by EGTA or Ca²⁺ channel blockers. A hyperpolarization-activated Ca²⁺-permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with whole-cell patch-clamp recording. Calmodulin (CaM) antiserum and W₇-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca²⁺]_c decrease, while exogenous purified CaM triggers a transient increase of [Ca²⁺]_c and also remarkably activated the hyperpolarization-activated Ca²⁺ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca²⁺]_c and the activation of Ca²⁺ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca²⁺ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca²⁺-permeable channel, whose activity may be regulated by extracellular CaM, in pollen cells.