The temperature-signaling cascade in sponges involves a heat-gated cation channel, abscisic acid, and cyclic ADP-ribose

Abstract

Sponges (phylum Porifera) are the phylogenetically oldest metazoan animals, their evolution dating back to 600 million years ago. Here we demonstrate that sponges express ADP-ribosyl cyclase activity, which converts NAD(+) into cyclic ADP-ribose, a potent and universal intracellular Ca(2+) mobilizer. In Axinella polypoides (Demospongiae, Axinellidae), ADP-ribosyl cyclase was activated by temperature increases by means of an abscisic acid-induced, protein kinase A-dependent mechanism. The thermosensor triggering this signaling cascade was a heat-activated cation channel. Elucidation of the complete thermosensing pathway in sponges highlights a number of features conserved in higher organisms: (i) the cation channel thermoreceptor, sensitive to heat, mechanical stress, phosphorylation, and anesthetics, shares all of the functional characteristics of the mammalian heat-activated background K(+) channel responsible for central and peripheral thermosensing; (ii) involvement of the phytohormone abscisic acid and cyclic ADP-ribose as its second messenger is reminiscent of the drought stress signaling pathway in plants. These results suggest an ancient evolutionary origin of this stress-signaling cascade in a common precursor of modern Metazoa and Metaphyta.

Figure 1

Effect of temperature, ABA, and AA on [Ca²⁺]_i in A. polypoides cells. Fura 2-loaded cells were incubated in CMF-SW, or SW, containing 10 mM glucose at the indicated temperatures in a thermostated microfluorimetric cuvette, under continuous slow stirring. The fluorescence emission ratio E340/E380 (R) is shown. Ratios were acquired every 0.5 s. Each point is the mean of 120 acquisitions. Traces were normalized to a zero-time R of 1.0 to facilitate comparison. (A) Temperature dependence of the R increase and effect of ABA. Incubations were performed in CMF-SW. ABA was added at time = zero. Traces are from one of five comparable experiments, performed on different animals. (B) Traces from two representative experiments showing the increase of the [Ca²⁺]_i as continuously recorded over 2-h exposure of the cells to 28°C or 28°C for 60 min, followed by 14°C for the rest of the recording time. (C) Inhibition of the temperature- and ABA-induced increase of [Ca²⁺]_i by 8-Br-cADPR and effect of AA on [Ca²⁺]_i. Fura 2-loaded cells were preincubated for 30 min at 14°C in SW with 20 μM 8-Br-cADPR, then washed, resuspended in SW, and exposed to 28°C in the cuvette. AA or palmitic acid (PA), both 50 μM, were added at time = zero to Fura-loaded cells at 14°C.

Figure 2

Patch-clamp recording of transmembrane currents in A. polypoides cells. (A) Voltage ramps of 5-s duration were applied from a holding potential of −20 mV in whole-cell configuration. Currents were recorded at 14°C and 26°C, as indicated. Sampling and filtering frequencies were 2 and 0.5 KHz, respectively. Cells were bathed with external artificial SW and internal IntA solution. The arrows show the monovalent cationic [V_rev(C⁺) caused by Na⁺ and K⁺] and anionic [V_rev(A⁻) caused by Cl⁻] reversal potentials, as calculated for the bathing and internal solutions in use. (Inset) The inhibition of the high temperature-induced current by 100 μM Gd³⁺. (B) Currents recorded before, during, and after application of AA (100 μM), at T = 14°C under the same experimental conditions as in A. (Inset) The inhibition of the AA-induced current by 300 μM La³⁺. (C) Single-channel recording of mechanical stress-activated channels in cell-attached configuration. Channel activity was recorded at atmospheric pressure and during stretch of −84 mmHg applied through the recording pipette (V_m = 20 mV), at 4-KHz sampling and 1-KHz filtering frequencies. Cells were bathed with external artificial SW and IntB solution into the pipette. (Inset) The single-channel I–V relationship and some example traces, measured at the minimum stretch needed to elicit the current. Each point has been evaluated from at least 50 single-channel transitions, and bars show the SEM. The channel conductance calculated from the best fit was 13 ± 4 pS.

Figure 3

The temperature-signaling cascade in A. polypoides.