Figure 6f: WT NeRh50 maximum amplitudes and decay rate constants for transient currents recorded in the presence of 200 mM NH₄⁺ under static pH (pH6.5), in the presence of an inward proton gradient (pH6.5 to 7.5), and in the absence of NH4+ under the same proton-gradient configuration

Manuscript figure 6d shows WT maximum amplitude and decay rate constant variation for transient currents recorded in the presence of 200 mM NH₄⁺ under static pH (pH6.5), in the presence of an inward proton gradient (pH6.5 to 7.5), and in the absence of NH4+ under the same proton-gradient configuration (figure 6f)

The Dataset

The data used for the figure are shown in Table 1

Show the code 
DT::datatable(dfms$dfm_6f, options=list(pageLength=5))
Table 1: Maximum amplitude and decay rate constant for transient currents recorded in the presence of 200 mM NH₄⁺ under static pH (pH6.5), in the presence of an inward proton gradient (pH6.5 to 7.5), and in the absence of NH4+ under the same proton-gradient configuration (figure 6f).

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The Models

We use linear models for amplitude and decay rate as dependent variables, with group as the independent factor, checking fit in Figure 1.

Show the code 
# Fit the models
model6f_amp_lm <- lm(amplitude ~ group, data=dfms$dfm_6f)
model6f_k_lm <- lm(decayrate ~ group, data=dfms$dfm_6f)

# Check model fit
performance::check_model(model6f_amp_lm)
performance::check_model(model6f_k_lm)
(a) amplitude ~ group
(b) decayrate ~ group
Figure 1: Diagnostic plots for linear model fits of amplitude ~ group and decayrate ~ group. There is some heteroscadicity in the maximum amplitude data, and there are suggestions of a slight departure from normality in the same data.

As the model fit is acceptable, we estimate contrasts in Table 2.

Show the code 
EMM <- emmeans::emmeans(model6f_amp_lm, ~ group)
confint(emmeans::contrast(EMM, "pairwise", adjust="sidak")) |>
  kableExtra::kbl(digits=3) |>
  kableExtra::kable_styling(
    bootstrap_options = c("hover", "responsive")
  )
EMM <- emmeans::emmeans(model6f_k_lm, ~ group)
confint(emmeans::contrast(EMM, "pairwise", adjust="sidak")) |>
  kableExtra::kbl(digits=3) |>
  kableExtra::kable_styling(
    bootstrap_options = c("hover", "responsive")
  )
Table 2: Contrasts for model fits, with Sidak adjustment. Not all contrasts include zero in the 95% confidence interval estimate.
(a) Maximum amplitude
contrast estimate SE df lower.CL upper.CL
pH6.5 (no gradient) - pH6.5>7.5 gradient NH4 0.153 0.122 17 -0.171 0.477
pH6.5 (no gradient) - pH6.5>7.5 gradient, no NH4 1.248 0.118 17 0.937 1.559
pH6.5>7.5 gradient NH4 - pH6.5>7.5 gradient, no NH4 1.095 0.122 17 0.771 1.419
(b) Decay rate
contrast estimate SE df lower.CL upper.CL
pH6.5 (no gradient) - pH6.5>7.5 gradient NH4 3.58 7.266 11 -12.411 19.571

For the maximum amplitude data we find that all contrasts with the pH6.5 to pH7.5 gradient with no NH4+ exclude zero from the 95% CI, but the contrast between pH6.5 and pH6.5 to pH7.5 with NH4+ indicates no difference between groups. There is also no support for a difference in decay rate constant between pH6.5 and pH6.5 to pH7.5 with NH4+.

The Figures

We plot the data, indicating P-values for the contrasts in Figure 2.

Show the code 
# Get p-values, accounting for heteroscedasticity and multiple testing
# Also generate group1 and group2 columns
EMM_amp <- as.data.frame(pairs(emmeans::emmeans(model6f_amp_lm, ~ group),
                               adjust="sidak")) |>
  tidyr::separate(contrast, into=c("group1", "group2"), sep=" - ") |>
  dplyr::mutate(y.position=2.5) |>
  rstatix::add_significance()

EMM_k <- as.data.frame(pairs(emmeans::emmeans(model6f_k_lm, ~ group),
                               adjust="sidak")) |>
  tidyr::separate(contrast, into=c("group1", "group2"), sep=" - ") |>
  dplyr::mutate(y.position=90) |>
  rstatix::add_significance()

p6f_amp <- ggplot2::ggplot(dfms$dfm_6f, 
                           ggplot2::aes(x=group, y=amplitude, color=group)) +
  ggplot2::geom_boxplot(outlier.shape = NA) +
  ggplot2::geom_jitter() +
  ggplot2::labs(y="Maximum amplitude (nA)", x="") +
  ggplot2::ylim(0, 4.5) +
  ggplot2::theme_minimal() +
  ggpubr::stat_pvalue_manual(EMM_amp,
                             label="p = {scales::pvalue(p.value)}",
                             size=3, step.increase=0.2)+
  ggplot2::theme(legend.position = "none",
                 axis.text.x = ggplot2::element_text(angle = 30, vjust = 0.5, hjust=1),
                 plot.margin=ggplot2::unit(c(0.5,0.5,1,0.5), 'cm'))

p6f_k <- ggplot2::ggplot(dfms$dfm_6f, 
                         ggplot2::aes(x=group, y=decayrate, color=group)) +
  ggplot2::geom_boxplot(outlier.shape = NA) +
  ggplot2::geom_jitter() +
  ggplot2::labs(y="Decay rate constant k (/s)", x="") +
  ggplot2::ylim(0, 105) +
  ggplot2::theme_minimal() +
  ggpubr::stat_pvalue_manual(EMM_k, 
                             label="p = {scales::pvalue(p.value)}", 
                             size=3, step.increase=0.15)+
  ggplot2::theme(legend.position = "none",
                 axis.text.x = ggplot2::element_text(angle = 30, vjust = 0.5, hjust=1),
                 plot.margin=ggplot2::unit(c(0.5,0.5,1,0.5), 'cm'))

p6f_amp
p6f_k

# Write figures to file (commented out for webpage)
#ggplot2::ggsave("assets/images/fig6f_WT_maximum_amplitude.pdf", p6f_amp)
#ggplot2::ggsave("assets/images/fig6f_WT_maximum_amplitude.png", p6f_amp)
#ggplot2::ggsave("assets/images/fig6f_WT_decay_rate_constant.png", p6f_k)
#ggplot2::ggsave("assets/images/fig6f_WT_decay_rate_constant.pdf", p6f_k)
(a) Figure 6f, maximum amplitude
(b) Figure 6f, decay rate
Figure 2: Maximum amplitude and decay rate constant for four pH regimes.

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