Rauh R et al. (2020), Effects of syntaxins 2, 3, and 4 on rat and hum...

XB-ART-56832

Pflugers Arch 2020 Apr 01;4724:461-471. doi: 10.1007/s00424-020-02365-6.

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Effects of syntaxins 2, 3, and 4 on rat and human epithelial sodium channel (ENaC) in Xenopus laevis oocytes.

Rauh R , Frost F , Korbmacher C .

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Syntaxins are SNARE proteins and may play a role in epithelial sodium channel (ENaC) trafficking. The aim of the present study was to investigate the effects of syntaxin 2 (STX2), syntaxin 3 (STX3), and syntaxin 4 (STX4) on rat (rENaC) and human ENaC (hENaC). Co-expression of rENaC and STX3 or STX4 in Xenopus laevis oocytes increased amiloride-sensitive whole-cell currents (ΔIami) on average by 50% and 135%, respectively, compared to oocytes expressing rENaC alone. In contrast, STX2 had no significant effect on rENaC. Similar to its effect on rENaC, STX3 stimulated hENaC by 48%. In contrast, STX2 and STX4 inhibited hENaC by 51% and 44%, respectively. Using rENaC carrying a FLAG tag in the extracellular loop of the β-subunit, we demonstrated that the stimulatory effects of STX3 and STX4 on ΔIami were associated with an increased expression of the channel at the cell surface. Co-expression of STX3 or STX4 did not significantly alter the degree of proteolytic channel activation by chymotrypsin. STX3 had no effect on the inhibition of rENaC by brefeldin A, and the stimulatory effect of STX3 was preserved in the presence of dominant negative Rab11. This indicates that the stimulatory effect of STX3 is not mediated by inhibiting channel retrieval or by stimulating fusion of recycling endosomes. Our results suggest that the effects of syntaxins on ENaC are isoform and species dependent. Furthermore, our results demonstrate that STX3 increases ENaC expression at the cell surface, probably by enhancing insertion of vesicles carrying newly synthesized channels.

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Species referenced: Xenopus laevis
Genes referenced: rab11a stx3

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	Figure 1. Syntaxins 3 and 4 stimulate rENaC function in oocytes. Oocytes were injected with cRNA for rENaC alone or together with different amounts of cRNA for syntaxin 2 (+STX2), syntaxin 3 (+STX3), or syntaxin 4 (+STX4) and incubated for 2 days. Amiloride-sensitive whole-cell currents (ΔIami) were measured with the two-electrode voltage-clamp technique. a, d, g Representative whole-cell current traces from matched control oocytes expressing rENaC alone or from oocytes co-expressing rENaC and a syntaxin isoform (1 ng cRNA), as indicated. The presence of amiloride (ami) in the bath solution is indicated by black bars. b, e, h Summary of data obtained from an individual batch of oocytes measured as shown in a, d, or g, respectively. c, f, i Summary of data as shown in b, e, and h obtained from several different batches of oocytes. To take batch-to-batch variability into account, individual ΔIami values were normalized to the mean ΔIami of the rENaC control group of the corresponding batch. Unpaired t test (b, e, h) or one-way ANOVA followed by Dunnett’s multiple comparison test vs. rENaC control (c, f, i), * p < 0.05, p < 0.01, * p < 0.001.
	Figure 2. Effects of syntaxins 2, 3, and 4 on hENaC function. Oocytes were injected with cRNA for hENaC alone or together with different amounts of cRNA for syntaxin 2 (+STX2, a), syntaxin 3 (+STX3, b), or syntaxin 4 (+STX4, c). To take batch-to-batch variability into account, individual ΔIami values were normalized to the mean ΔIami of the hENaC control group of the corresponding batch. One-way ANOVA followed by Dunnett’s multiple comparison test vs. hENaC control, p < 0.05, p < 0.01, **p < 0.001
	Figure 3. Syntaxins 3 and 4 increase surface expression of rENaC. ΔIami (a) and surface expression (b) were measured in parallel in oocytes expressing rENaC carrying a FLAG reporter epitope in the extracellular domain of the β-subunit (rENaCFLAG) alone or together with syntaxin 3 (+STX3) or syntaxin 4 (+STX4). Oocytes injected with twice the amount of cRNA for rENaCFLAG (rENaCFLAG 2×) served as positive control. In three batches of oocytes, oocytes were injected with 0.025 ng cRNA/subunit for rENaCFLAG and with 1 ng cRNA for syntaxin 3 or syntaxin 4. In five batches of oocytes, oocytes were injected with 0.05 ng cRNA/subunit for rENaCFLAG and with 2 ng cRNA for syntaxin 3 or syntaxin 4. To summarize data from different batches of oocytes, values were normalized to the mean of the corresponding rENaCFLAG control group. One-way ANOVA followed by Dunnett’s multiple comparison test vs. rENaCFLAG control, p < 0.01, *p < 0.001
	Figure 4. Syntaxins 3 and 4 do not affect proteolytic activation of rENaC by chymotrypsin. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for syntaxin 3 (STX3, 1 ng) or syntaxin 4 (STX4, 1 ng) and incubated for 2 days. a, d Typical whole-cell current traces of oocytes expressing rENaC alone or together with syntaxin 3 (a) or syntaxin 4 (d). b, e Summary of ΔIami values before and after activation of rENaC with chymotrypsin (2 μg/ml) obtained from similar experiments as shown in a and d, respectively. ΔIami values before and after application of chymotrypsin were compared with paired t test; corresponding ΔIami values in the absence and presence of syntaxin 3 or syntaxin 4 were compared with unpaired t test. c, f Ratio of ΔIami before (ΔIami-initial) and after (ΔIami-chymo) activation of rENaC with chymotrypsin was calculated from individual oocytes as a measure of average channel Po; unpaired t test. n.s. not significant, p < 0.01, *p < 0.001
	Figure 5. Syntaxin 3 stimulates rENaC insertion into the plasma membrane. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for syntaxin 3 (1 ng) and incubated for 2 days. After the initial measurement of ΔIami at 0 h, half of the oocytes were incubated for 8 h in the presence of brefeldin A (BFA, 5 μM), as indicated, and ΔIami was measured at 4, 8, and 24 h. Each data point represents ΔIami values from 28 to 30 oocytes. For better comparison, ΔIami was normalized to the rENaC expressing control group
	Figure 6. Blocking endosomal recycling does not prevent the stimulation of rENaC by syntaxin 3. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for dominant-negative Rab11a (5 ng, +dnRab11a), syntaxin 3 (1 ng, +STX3), or both. ΔIami was measured after 2 days of incubation. One-way ANOVA followed by Bonferroni’s multiple comparison test with selected pairs, p < 0.05, p < 0.01, **p < 0.001
	Fig. 1. Syntaxins 3 and 4 stimulate rENaC function in oocytes. Oocytes were injected with cRNA for rENaC alone or together with different amounts of cRNA for syntaxin 2 (+STX2), syntaxin 3 (+STX3), or syntaxin 4 (+STX4) and incubated for 2 days. Amiloride-sensitive whole-cell currents (ΔIami) were measured with the two-electrode voltage-clamp technique. a, d, g Representative whole-cell current traces from matched control oocytes expressing rENaC alone or from oocytes co-expressing rENaC and a syntaxin isoform (1 ng cRNA), as indicated. The presence of amiloride (ami) in the bath solution is indicated by black bars. b, e, h Summary of data obtained from an individual batch of oocytes measured as shown in a, d, or g, respectively. c, f, i Summary of data as shown in b, e, and h obtained from several different batches of oocytes. To take batch-to-batch variability into account, individual ΔIami values were normalized to the mean ΔIami of the rENaC control group of the corresponding batch. Unpaired t test (b, e, h) or one-way ANOVA followed by Dunnett’s multiple comparison test vs. rENaC control (c, f, i), * p < 0.05, p < 0.01, * p < 0.001
	Fig. 2. Effects of syntaxins 2, 3, and 4 on hENaC function. Oocytes were injected with cRNA for hENaC alone or together with different amounts of cRNA for syntaxin 2 (+STX2, a), syntaxin 3 (+STX3, b), or syntaxin 4 (+STX4, c). To take batch-to-batch variability into account, individual ΔIami values were normalized to the mean ΔIami of the hENaC control group of the corresponding batch. One-way ANOVA followed by Dunnett’s multiple comparison test vs. hENaC control, p < 0.05, p < 0.01, **p < 0.001
	Fig. 3. Syntaxins 3 and 4 increase surface expression of rENaC. ΔIami (a) and surface expression (b) were measured in parallel in oocytes expressing rENaC carrying a FLAG reporter epitope in the extracellular domain of the β-subunit (rENaCFLAG) alone or together with syntaxin 3 (+STX3) or syntaxin 4 (+STX4). Oocytes injected with twice the amount of cRNA for rENaCFLAG (rENaCFLAG 2×) served as positive control. In three batches of oocytes, oocytes were injected with 0.025 ng cRNA/subunit for rENaCFLAG and with 1 ng cRNA for syntaxin 3 or syntaxin 4. In five batches of oocytes, oocytes were injected with 0.05 ng cRNA/subunit for rENaCFLAG and with 2 ng cRNA for syntaxin 3 or syntaxin 4. To summarize data from different batches of oocytes, values were normalized to the mean of the corresponding rENaCFLAG control group. One-way ANOVA followed by Dunnett’s multiple comparison test vs. rENaCFLAG control, p < 0.01, *p < 0.001
	Fig. 4. Syntaxins 3 and 4 do not affect proteolytic activation of rENaC by chymotrypsin. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for syntaxin 3 (STX3, 1 ng) or syntaxin 4 (STX4, 1 ng) and incubated for 2 days. a, d Typical whole-cell current traces of oocytes expressing rENaC alone or together with syntaxin 3 (a) or syntaxin 4 (d). b, e Summary of ΔIami values before and after activation of rENaC with chymotrypsin (2 μg/ml) obtained from similar experiments as shown in a and d, respectively. ΔIami values before and after application of chymotrypsin were compared with paired t test; corresponding ΔIami values in the absence and presence of syntaxin 3 or syntaxin 4 were compared with unpaired t test. c, f Ratio of ΔIami before (ΔIami-initial) and after (ΔIami-chymo) activation of rENaC with chymotrypsin was calculated from individual oocytes as a measure of average channel Po; unpaired t test. n.s. not significant, p < 0.01, *p < 0.001
	Fig. 5. Syntaxin 3 stimulates rENaC insertion into the plasma membrane. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for syntaxin 3 (1 ng) and incubated for 2 days. After the initial measurement of ΔIami at 0 h, half of the oocytes were incubated for 8 h in the presence of brefeldin A (BFA, 5 μM), as indicated, and ΔIami was measured at 4, 8, and 24 h. Each data point represents ΔIami values from 28 to 30 oocytes. For better comparison, ΔIami was normalized to the rENaC expressing control group
	Fig. 6. Blocking endosomal recycling does not prevent the stimulation of rENaC by syntaxin 3. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for dominant-negative Rab11a (5 ng, +dnRab11a), syntaxin 3 (1 ng, +STX3), or both. ΔIami was measured after 2 days of incubation. One-way ANOVA followed by Bonferroni’s multiple comparison test with selected pairs, p < 0.05, p < 0.01, **p < 0.001

References [+] :

Anantharam, Open probability of the epithelial sodium channel is regulated by intracellular sodium. 2006, Pubmed, Xenbase

	Figure 1. Syntaxins 3 and 4 stimulate rENaC function in oocytes. Oocytes were injected with cRNA for rENaC alone or together with different amounts of cRNA for syntaxin 2 (+STX2), syntaxin 3 (+STX3), or syntaxin 4 (+STX4) and incubated for 2 days. Amiloride-sensitive whole-cell currents (ΔIami) were measured with the two-electrode voltage-clamp technique. a, d, g Representative whole-cell current traces from matched control oocytes expressing rENaC alone or from oocytes co-expressing rENaC and a syntaxin isoform (1 ng cRNA), as indicated. The presence of amiloride (ami) in the bath solution is indicated by black bars. b, e, h Summary of data obtained from an individual batch of oocytes measured as shown in a, d, or g, respectively. c, f, i Summary of data as shown in b, e, and h obtained from several different batches of oocytes. To take batch-to-batch variability into account, individual ΔIami values were normalized to the mean ΔIami of the rENaC control group of the corresponding batch. Unpaired t test (b, e, h) or one-way ANOVA followed by Dunnett’s multiple comparison test vs. rENaC control (c, f, i), * p < 0.05, p < 0.01, * p < 0.001.
	Figure 2. Effects of syntaxins 2, 3, and 4 on hENaC function. Oocytes were injected with cRNA for hENaC alone or together with different amounts of cRNA for syntaxin 2 (+STX2, a), syntaxin 3 (+STX3, b), or syntaxin 4 (+STX4, c). To take batch-to-batch variability into account, individual ΔIami values were normalized to the mean ΔIami of the hENaC control group of the corresponding batch. One-way ANOVA followed by Dunnett’s multiple comparison test vs. hENaC control, p < 0.05, p < 0.01, **p < 0.001
	Figure 3. Syntaxins 3 and 4 increase surface expression of rENaC. ΔIami (a) and surface expression (b) were measured in parallel in oocytes expressing rENaC carrying a FLAG reporter epitope in the extracellular domain of the β-subunit (rENaCFLAG) alone or together with syntaxin 3 (+STX3) or syntaxin 4 (+STX4). Oocytes injected with twice the amount of cRNA for rENaCFLAG (rENaCFLAG 2×) served as positive control. In three batches of oocytes, oocytes were injected with 0.025 ng cRNA/subunit for rENaCFLAG and with 1 ng cRNA for syntaxin 3 or syntaxin 4. In five batches of oocytes, oocytes were injected with 0.05 ng cRNA/subunit for rENaCFLAG and with 2 ng cRNA for syntaxin 3 or syntaxin 4. To summarize data from different batches of oocytes, values were normalized to the mean of the corresponding rENaCFLAG control group. One-way ANOVA followed by Dunnett’s multiple comparison test vs. rENaCFLAG control, p < 0.01, *p < 0.001
	Figure 4. Syntaxins 3 and 4 do not affect proteolytic activation of rENaC by chymotrypsin. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for syntaxin 3 (STX3, 1 ng) or syntaxin 4 (STX4, 1 ng) and incubated for 2 days. a, d Typical whole-cell current traces of oocytes expressing rENaC alone or together with syntaxin 3 (a) or syntaxin 4 (d). b, e Summary of ΔIami values before and after activation of rENaC with chymotrypsin (2 μg/ml) obtained from similar experiments as shown in a and d, respectively. ΔIami values before and after application of chymotrypsin were compared with paired t test; corresponding ΔIami values in the absence and presence of syntaxin 3 or syntaxin 4 were compared with unpaired t test. c, f Ratio of ΔIami before (ΔIami-initial) and after (ΔIami-chymo) activation of rENaC with chymotrypsin was calculated from individual oocytes as a measure of average channel Po; unpaired t test. n.s. not significant, p < 0.01, *p < 0.001
	Figure 5. Syntaxin 3 stimulates rENaC insertion into the plasma membrane. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for syntaxin 3 (1 ng) and incubated for 2 days. After the initial measurement of ΔIami at 0 h, half of the oocytes were incubated for 8 h in the presence of brefeldin A (BFA, 5 μM), as indicated, and ΔIami was measured at 4, 8, and 24 h. Each data point represents ΔIami values from 28 to 30 oocytes. For better comparison, ΔIami was normalized to the rENaC expressing control group
	Figure 6. Blocking endosomal recycling does not prevent the stimulation of rENaC by syntaxin 3. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for dominant-negative Rab11a (5 ng, +dnRab11a), syntaxin 3 (1 ng, +STX3), or both. ΔIami was measured after 2 days of incubation. One-way ANOVA followed by Bonferroni’s multiple comparison test with selected pairs, p < 0.05, p < 0.01, **p < 0.001
	Fig. 1. Syntaxins 3 and 4 stimulate rENaC function in oocytes. Oocytes were injected with cRNA for rENaC alone or together with different amounts of cRNA for syntaxin 2 (+STX2), syntaxin 3 (+STX3), or syntaxin 4 (+STX4) and incubated for 2 days. Amiloride-sensitive whole-cell currents (ΔIami) were measured with the two-electrode voltage-clamp technique. a, d, g Representative whole-cell current traces from matched control oocytes expressing rENaC alone or from oocytes co-expressing rENaC and a syntaxin isoform (1 ng cRNA), as indicated. The presence of amiloride (ami) in the bath solution is indicated by black bars. b, e, h Summary of data obtained from an individual batch of oocytes measured as shown in a, d, or g, respectively. c, f, i Summary of data as shown in b, e, and h obtained from several different batches of oocytes. To take batch-to-batch variability into account, individual ΔIami values were normalized to the mean ΔIami of the rENaC control group of the corresponding batch. Unpaired t test (b, e, h) or one-way ANOVA followed by Dunnett’s multiple comparison test vs. rENaC control (c, f, i), * p < 0.05, p < 0.01, * p < 0.001
	Fig. 2. Effects of syntaxins 2, 3, and 4 on hENaC function. Oocytes were injected with cRNA for hENaC alone or together with different amounts of cRNA for syntaxin 2 (+STX2, a), syntaxin 3 (+STX3, b), or syntaxin 4 (+STX4, c). To take batch-to-batch variability into account, individual ΔIami values were normalized to the mean ΔIami of the hENaC control group of the corresponding batch. One-way ANOVA followed by Dunnett’s multiple comparison test vs. hENaC control, p < 0.05, p < 0.01, **p < 0.001
	Fig. 3. Syntaxins 3 and 4 increase surface expression of rENaC. ΔIami (a) and surface expression (b) were measured in parallel in oocytes expressing rENaC carrying a FLAG reporter epitope in the extracellular domain of the β-subunit (rENaCFLAG) alone or together with syntaxin 3 (+STX3) or syntaxin 4 (+STX4). Oocytes injected with twice the amount of cRNA for rENaCFLAG (rENaCFLAG 2×) served as positive control. In three batches of oocytes, oocytes were injected with 0.025 ng cRNA/subunit for rENaCFLAG and with 1 ng cRNA for syntaxin 3 or syntaxin 4. In five batches of oocytes, oocytes were injected with 0.05 ng cRNA/subunit for rENaCFLAG and with 2 ng cRNA for syntaxin 3 or syntaxin 4. To summarize data from different batches of oocytes, values were normalized to the mean of the corresponding rENaCFLAG control group. One-way ANOVA followed by Dunnett’s multiple comparison test vs. rENaCFLAG control, p < 0.01, *p < 0.001
	Fig. 4. Syntaxins 3 and 4 do not affect proteolytic activation of rENaC by chymotrypsin. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for syntaxin 3 (STX3, 1 ng) or syntaxin 4 (STX4, 1 ng) and incubated for 2 days. a, d Typical whole-cell current traces of oocytes expressing rENaC alone or together with syntaxin 3 (a) or syntaxin 4 (d). b, e Summary of ΔIami values before and after activation of rENaC with chymotrypsin (2 μg/ml) obtained from similar experiments as shown in a and d, respectively. ΔIami values before and after application of chymotrypsin were compared with paired t test; corresponding ΔIami values in the absence and presence of syntaxin 3 or syntaxin 4 were compared with unpaired t test. c, f Ratio of ΔIami before (ΔIami-initial) and after (ΔIami-chymo) activation of rENaC with chymotrypsin was calculated from individual oocytes as a measure of average channel Po; unpaired t test. n.s. not significant, p < 0.01, *p < 0.001
	Fig. 5. Syntaxin 3 stimulates rENaC insertion into the plasma membrane. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for syntaxin 3 (1 ng) and incubated for 2 days. After the initial measurement of ΔIami at 0 h, half of the oocytes were incubated for 8 h in the presence of brefeldin A (BFA, 5 μM), as indicated, and ΔIami was measured at 4, 8, and 24 h. Each data point represents ΔIami values from 28 to 30 oocytes. For better comparison, ΔIami was normalized to the rENaC expressing control group
	Fig. 6. Blocking endosomal recycling does not prevent the stimulation of rENaC by syntaxin 3. Oocytes were injected with cRNA for rENaC (0.025 ng/subunit) alone or together with cRNA for dominant-negative Rab11a (5 ng, +dnRab11a), syntaxin 3 (1 ng, +STX3), or both. ΔIami was measured after 2 days of incubation. One-way ANOVA followed by Bonferroni’s multiple comparison test with selected pairs, p < 0.05, p < 0.01, **p < 0.001