Dürrnagel S et al. (2012), High Ca(2+) permeability of a peptide-gated DEG...

XB-ART-45983

J Gen Physiol 2012 Oct 01;1404:391-402. doi: 10.1085/jgp.201210798.

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High Ca(2+) permeability of a peptide-gated DEG/ENaC from Hydra.

Dürrnagel S , Falkenburger BH , Gründer S .

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Degenerin/epithelial Na(+) channels (DEG/ENaCs) are Na(+) channels that are blocked by the diuretic amiloride. In general, they are impermeable for Ca(2+) or have a very low permeability for Ca(2+). We describe here, however, that a DEG/ENaC from the cnidarian Hydra magnipapillata, the Hydra Na(+) channel (HyNaC), is highly permeable for Ca(2+) (P(Ca)/P(Na) = 3.8). HyNaC is directly gated by Hydra neuropeptides, and in Xenopus laevis oocytes expressing HyNaCs, RFamides elicit currents with biphasic kinetics, with a fast transient component and a slower sustained component. Although it was previously reported that the sustained component is unselective for monovalent cations, the selectivity of the transient component had remained unknown. Here, we show that the transient current component arises from secondary activation of the Ca(2+)-activated Cl(-) channel (CaCC) of Xenopus oocytes. Inhibiting the activation of the CaCC leads to a simple on-off response of peptide-activated currents with no apparent desensitization. In addition, we identify a conserved ring of negative charges at the outer entrance of the HyNaC pore that is crucial for the high Ca(2+) permeability, presumably by attracting divalent cations to the pore. At more positive membrane potentials, the binding of Ca(2+) to the ring of negative charges increasingly blocks HyNaC currents. Thus, HyNaC is the first member of the DEG/ENaC gene family with a high Ca(2+) permeability.

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Species referenced: Xenopus laevis
Genes referenced: asic1 clca1.3 nppb p2rx4 tbx2

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	Figure 1. Transient peptide-activated currents depend on Cl−. (A) Representative current trace showing activation of HyNaC2/3/5 in standard bath. HyNaC was activated with 1 µM Hydra-RFamide I (black bars). (B) Repeated activation of HyNaC at different holding potentials (Vhold) reveals the different Erevs of the transient and the sustained current component. A representative current trace in standard bath solution is shown. Note that the transient current changes to outward at more negative potentials than the sustained current. The inset highlights the three current phases (initial inward, transient outward, and sustained inward) at a holding potential of −20 mV. (C) I/V plot revealing the different Erevs of the transient (open squares) and sustained current components (closed squares) in standard bath (n = 6). Note the strong inward rectification of both components. The inset shows the I/V relations close to the Erevs on an expanded scale. (D) Representative current trace in bath solution lacking Cl− (NaCl replaced by NaGluconate). Note the absence of transient outward currents.
	Figure 2. Chelation of [Ca2+]i abolishes transient peptide-activated currents. (A; top) Representative HyNaC2/3/5 currents in oocytes injected with EGTA or BAPTA. Note the absence of a transient current and the step-like appearance of the currents. (Bottom) Bar graphs comparing the peak (hatched bars) and sustained (open bars) current amplitudes of oocytes expressing HyNaC and of oocytes expressing HyNaC and injected with EGTA (closed bars) or BAPTA (gray bars) (n = 11). (B; top) Representative current trace comparing peptide-activated currents in standard bath and in standard bath in which CaCl2 was replaced by an equimolar amount (1.8 mM) of BaCl2. Currents were measured either in the presence or the absence of 100 µM NPPB. Oocytes were preincubated for 20 s in the corresponding test solutions before activation by 1 µM RFamide I. (Bottom) Bar graph comparing the current amplitudes in the absence of NPPB (open bars) and in the presence of 100 µM NPPB (gray bars) for the indicated conditions. In the presence of Ba2+, no transient current was discernible. The application sequence of the individual solutions was shuffled (n = 8). (C) As in B, for oocytes that had been injected with EGTA. Note the absence of peak currents. (Bottom) Bar graph comparing the current amplitudes in the absence of NPPB (closed bars) and in the presence of 100 µM NPPB (gray bars) for sustained currents. , P < 0.05; , P < 0.01; **, P < 0.001.
	Figure 3. Peptide-activated currents in the absence of Na+e. (A; left) Representative current trace for HyNaC2/3/5 in solutions containing 1 mM Ca2+ together with 140 mM of either NMDG+ or Na+. 1 µM RFamide I was used for activation. (Right) Control experiment in NMDG+ solution nominally free of Ca2+. Similar results were observed in four out of four oocytes. (B; left) Representative current trace for HyNaC2/3/5 in solutions containing 10 mM Ca2+, 10 mM Ca2+ and 10 mM Na+, or 10 mM Na+ (nominally free of Ca2+). Solutions additionally contained 10 mM HEPES, pH 7.4, and NMDG+ at concentrations to reach similar osmolarity (10 Ca2+: 125 mM NMDGCl; 10 Ca2+/10 Na+: 115 mM NMDGCl; and 10 Na+: 130 mM NMDGCl). Oocytes had been injected with EGTA; 2 µM RFamide I was used for activation. Holding potential was −85 mV. (Right) Quantitative comparison of current amplitudes. ***, P < 0.001.
	Figure 4. Preventing a rise in [Ca2+]i changes Erev of sustained peptide-activated currents. I/V relations for HyNaC2/3/5 activated by 1 µM Hydra-RFamide I in standard bath (open circles), in standard bath after injection of EGTA (closed squares), and in standard bath in which CaCl2 had been replaced by BaCl2 (closed triangles). Currents were normalized to the current measured at −70 mV, which had an amplitude of −4.4 ± 1.1 µA (Ca2+), −1.4 ± 0.2 µA (Ca2+/EGTA), or −6.0 ± 0.8 µA (Ba2+) (n = 7 - 8), respectively. The inset shows the I/V relations close to the Erevs on an expanded scale.
	Figure 5. Activation of HyNaC increases [Ca2+]i in oocytes. (A) Representative trace (black) from photometric Ca2+ measurements illustrating the increase of the F340/380 ratio in a HyNaC2/3/5-expressing oocyte after the application of RFamide I (asterisk). Oocytes had been injected with 50 nl Fura-2AM (1 mM) 30–120 min before the recording. Thus, the increased F340/380 ratio reflects an increase in [Ca2+]i. The red trace represents the current that paralleled the rise in [Ca2+]i. HyNaC was activated by pipetting Hydra-RFamide I into the bath to yield a final concentration of ∼1 µM RFamide I. (B) Changes of the F340/F380 ratio caused by activation of HyNaC for individual measurements (n = 8).
	Figure 6. Permeability ratio PCa/PNa of HyNaC is comparable to P2X4. (A) I/V relations of HyNaC2/3/5 in solutions containing 1 or 10 mM CaCl2 and no NaCl (which had been replaced by 140 or 126.5 mM NMDG-Cl, respectively) and in a solution containing 140 mM NaCl and 1 mM CaCl2. Continuous voltage ramps were run with a speed of 0.46 ms per millivolt (thus, for example, 6 s per 130 mV). Different voltage ranges were chosen for different conditions. Currents were normalized to Imax. Lines are the sum of 8–10 individual measurements. SEM is indicated by gray errors bars. 50 nl EGTA (20 mM) was injected before the recordings. Squares represent the Erevs that are plotted in B. HyNaC was activated by 2 µM RFamide I. (B) Ca2+-dependent shifts of the Erev reveal a high Ca2+ permeability of HyNaC. Erevs of the highly Ca2+-permeable P2X4 receptor are shown for comparison and were similar to HyNaC. P2X4 was activated by 10 µM ATP.
	Figure 7. A conserved aspartate is necessary for the high Ca2+ permeability of HyNaC. (A) Alignment of the amino acid sequences of HyNaC2, 3, and 5 and ASIC1a at the beginning of the second transmembrane domain (TMD2) in the one-letter code. The crucial aspartate is indicated by an asterisk. Completely conserved amino acids are shown as white letters on a black background. (B) Representative current trace for HyNaC_D-C activated by 1 µM RFamide I. The amplitude and shape of the transient current component were more variable than for the wt. Mean maximum current amplitudes were 3.45 ± 1.2 µA for the first activation (n = 5). (C) Representative current trace showing activation of HyNaC_D-C by 1 µM RFamide I in solutions containing 1 mM Ca2+ together with either 140 mM NMDG+ or Na+. No inward current was visible in NMDG+ solutions. (D) I/V plot for HyNaC_D-C activated by 1 µM RFamide I in standard bath solution. Currents were normalized to the current measured at −70 mV, which had an amplitude of −1.1 ± 0.2 µA (n = 6). (E; left) Representative current trace for HyNaC_D-C in solutions containing 10 mM Ca2+, 10 mM Ca2+ and 10 mM Na+, or 10 mM Na+ (nominally free of Ca2+). Conditions were as in Fig. 3 B. (Right) Quantitative comparison of current amplitudes. ***, P < 0.001. (F) I/V relations of HyNaC_D-C in solutions containing 1 or 10 mM CaCl2 and no NaCl (which had been replaced by 140 or 126.5 mM NMDG-Cl, respectively) and in a solution containing 140 mM NaCl and 1 mM CaCl2. Lines are the sum of eight to nine individual measurements. SEM is indicated by gray errors bars. Experimental conditions were as in Fig. 6 A. Triangles represent the Erevs that are plotted in G. (G) Ca2+-dependent shifts of the Erev reveal a significantly lower Ca2+ permeability of HyNaC_D-C compared with HyNaC wt. Erevs of HyNaC wt and P2X4 (gray symbols) are from Fig. 6 B and shown for comparison. (H) Representative trace (black) from photometric Ca2+ measurements illustrating no change of the F340/380 ratio after the application of ∼1 µM RFamide I (asterisk) in a oocyte expressing HyNaC_D-C. The deflections in the F340/380 ratio immediately after the peptide was added are caused by movements of the solution and are unspecific. Oocytes had been injected with 50 nl Fura-2AM (1 mM) 30–120 min before the recording. The red trace represents the current that robustly increased after peptide application. (I) F340/F380 before and after the application of ∼1 µM RFamide I for HyNaC_D-C (n = 8). (J) Plot of the increase of the F340/380 ratio (as percentage) after the application of RFamide I as a function of peptide-activated current amplitudes. Closed squares represent individual measurements for HyNaC wt, and open triangles are for HyNaC_D-C (n = 8). Current amplitudes were smaller for HyNaC_D-C than for HyNaC wt (n = 8; P < 0.001; Fig. 5 B). Solid lines represent linear fits of the fluorescence/current ratio. Note that the slope of this line was larger for wt than for the D-C mutant, indicating a higher relative Ca2+ permeability of the wt.
	Figure 8. Scheme illustrating Ca2+ permeation through HyNaC. In the closed conformation (left), Ca2+ can probably access the extracellular vestibule through three lateral fenestrations (Jasti et al., 2007; Li et al., 2011) and bind to the conserved aspartates at the outer mouth of the ion pore. After binding of the ligand, HyNaC opens and, at negative membrane potentials, Ca2+ permeates the channel (−70 mV, middle), liberating the ion pore. At depolarized potentials (+30 mV, right), however, Ca2+ remains bound to the ring of negative charges and blocks the open pore. Based on the crystal structure of chicken ASIC1 (Jasti et al., 2007).

References [+] :

Barish, A transient calcium-dependent chloride current in the immature Xenopus oocyte. 1983, Pubmed, Xenbase