Identification and Characterization of CNGC18 as a Ca2+-Permeable Divalent Cation-Selective Channel in Arabidopsis Pollen Tubes

Extracellular Ca²⁺ influx focusing at the tips of pollen tubes is the main source of Ca²⁺ for the pollen tube tip cytosolic Ca²⁺ gradient, and plasma membrane Ca²⁺ channels were proposed to be presence in the tips of pollen tubes. It has been reported that the knockout mutation of CNGC18 led to a lethal male sterile phenotype, and CNGC18 was suggested as an essential Ca²⁺ channel candidate. However, the identity and characterization of CNGC18 as a Ca²⁺ channel remain to be addressed. Using patch clamping technique, we recorded obvious inward currents in HEK293T cells expressing CNGC18, and just a small background conductance was observed in mock cells. The removal of external Ca²⁺ abolished the CNGC18-mediated currents, and the increasing and decreasing of external Ca²⁺ concentration resulted in a shift of reversal potential from 0 mV to a more positive and a more negative voltages, respectively. Similar large inward currents were observed under the a substitution of external Ca²⁺ by Ba²⁺ in the same concentration. Two well-known Ca²⁺ channel blockers, La³⁺ (100 uM) and Gd³⁺ (100 uM), inhibited CNGC18-mediated inward currents dramatically. Furthermore, we did not observe any detectable inward currents in HEK293T cells expressing CNGC18 using K⁺ bath solution in which Ca²⁺ was replaced with K⁺, but  large inward K⁺ currents were observed in HEK293T cells expressing KAT1, a well-known K⁺ channel encoding gene. Together, these data clearly showed that CNGC18 was a Ca²⁺-permeable divalent cation-selective channel with no or a very low permeability to monovalent cations.