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The Arabidopsis cyclic nucleotide-gated channel (CNGC) family consists of 20 members, which have been reported to participate in various physiological processes, such as pathogen defense, development, and thermotolerance. Although CNGC11 and CNGC12 have been identified a decade ago and their role in programmed cell death is well studied, their precise channel regulation has not been studied electrophysiologically. Here, we determined the channel activities of CNGC11 and CNGC12 utilizing the two-electrode voltage-clamp technique in the Xenopus laevis oocyte heterologous expression system. Our results suggest that CNGC12 but not CNGC11 functions as an active calcium channel. Furthermore, the cyclic nucleotide monophosphates (cNMPs) did not affect the activities of CNGC11 nor CNGC12 in Xenopus oocytes. Interestingly, while the activity of CNGC11 was not affected by co-expression with calmodulin (CaM), the activity of CNGC12 was significantly enhanced when CaM1 was co-expressed in oocytes. This study reveals that the channel activities and the mechanisms of regulation by CaM are different between CNGC11 and CNGC12.
Figure 1. CNGC12 is a Ca2+-permeable, divalent cation-selective channel. (A) Typical whole-cell currents recorded from CNGC12- or CNGC11-expressing oocytes in bath solution containing 30 mM Ca2+ (upper set) or 30 mM Mg2+ (lower set) at pH 5.5; the water-injected oocytes were used as control. (B) Current–voltage (I–V) curves based on data from (A). The data are expressed as means ± SE, with n = 5 for each group. (C) Ion selectivity of CNGC12 and CNGC11. The currents generated by CNGC12 and CNGC11 at −140 mV, when oocytes were perfused with a solution containing 30 mM Ca2+, 30 mM Mg2+, 30 mM Ba2+, 30 mM K+, or 30 mM Na+, pH = 5.5. The data are expressed as means ± SE, with n = 6 for each group. (D) Whole-cell currents recorded from CNGC12-expressing oocytes perfused with the bath solution containing different concentrations of CaCl2 (10, 20, or 30 mM). (E) Current–voltage (I–V) curves recorded as in (D). The data are expressed as means ± SE, 10 mM, n = 4; 20 mM, n = 4; or 30 mM, n = 6. (F) Whole-cell currents recorded CNGC12-expressing oocytes when the oocytes are in a bath solution containing 30 mM Ca2+ and 100 μM LaCl3/100 μM GdCl3. (G) Current–voltage (I–V) curves recorded as in (E). The data are expressed as means ± SE, with n = 5 for each group. (H) The inward currents were greatly inhibited by bath solution containing 30 mM Ca2+ and 100 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS).
Figure 2. Cyclic nucleotide monophosphates (cNMPs) do not affect the activities of CNGC11 and CNGC12 in the Xenopus oocytes. (A) Typical current traces recorded in oocytes expressing water (control), CNGC12, CNGC11, CNGC12 plus CNGC11 (CNGC12 + CNGC11). The oocytes were perfused with a bath solution containing 30 mM Ca2+ (left), a bath solution containing 30 mM Ca2+ and 0.1 mM dibutyryl-cyclic AMP (cAMP) (middle), or the membrane-permeable cGMP analog 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP) (right). (B) Quantitative analysis of currents at −140 mV as in (A). Values are expressed as mean ± SE, with n = 6 for each group.
Figure 3. CaM1 physically interacts with CNGC12. (A) Yeast two-hybrid assay of calmodulins (CaMs)/CaM-like proteins (CMLs) and the C-terminal of CNGC12 (CNGC12-CT) and CNGC11 (CNGC11-CT). The combination of pGBKT7-CNGC12-CT/pGBKT7-CNGC11-CT and empty pGADT7 vectors were used as a negative control. SD/−Trp/−Leu represents synthetic dextrose minimal medium without tryptophan and leucine; SD/−Trp/−Leu/−His indicates synthetic dextrose minimal medium without tryptophan, leucine, and histidine. Growth of yeast on SD/−Trp/−Leu/−His plate indicates interaction between the two tested proteins. (B) Bimolecular fluorescence complementation (BiFC) analysis between CNGC12 and CaM1 or CNGC11 in Arabidopsis mesophyll protoplasts. Vectors encoding CNGC12-nVenus, CaM1-cCFP, CNGC11-cCFP, and nVenus were co-expressed in various combinations in Arabidopsis protoplasts. Scale bars, 5 μm. (C) Diagram representing various domains of the C-terminus of CNGC12 (CNGC12-CT) which were cloned and used as prey during yeast two-hybrid screening: 441D-650* (the C-terminal of CNGC12); 550R-650* [the C-terminal of CNGC12 without the first cyclic nucleotide-binding domain (CNBD)]; 565R-650* (the C-terminal of CNGC12 without two CNBDs); 565R-594P (IQ domain); 594P-650* (after the IQ domain to the end of CNGC12). (D) Yeast two-hybrid analysis of CaM1 with various fragments of CNGC12-CT.
Figure 4. CaM1 activates CNGC12 in Xenopus oocytes. (A) Whole-cell currents were recorded from the oocytes injected with water (control), CNGC12, CNGC12 plus CaM1 (CNGC12 + CaM1), CNGC12 plus CaM11234 (CNGC12 + CaM11234), CNGC12 plus CaM6 (CNGC12 + CaM6), CNGC11, CNGC1 plus CNGC12 and CaM1 (CNGC11 + CNGC12 + CaM1), CNGC11 plus CaM1 (CNGC11 + CaM1), CNGC11 plus CaM11234 (CNGC11 + CaM11234), and CNGC11 and CaM6 (CNGC11 + CaM6). The oocytes were perfused with bath solution containing 30 mM Ca2+ (left). (B) Effects of CaM1, CaM11234, and CNGC11 on the currents generated by CNGC12 at −140 mV. The pooled current values were at 1.8 s of each voltage-clamp episode. The data are expressed as means ± SE. (water, n = 6; CNGC12, n = 6; CNGC12 + CaM1, n = 8; CNGC12 + CaM11234, n = 5; CNGC12 + CaM6, n = 7; CNGC11, n = 6; CNGC11 + CNGC12 + CaM1, n = 6; CNGC11 + CaM1, n = 6; CNGC11 + CaM11234, n = 6; and CNGC11 + CaM6, n = 8).
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