Figure 5a: WT NeRh50 maximum amplitudes and decay rate constants in response to changes in ligand

Manuscript figure 5a shows WT maximum amplitude and decay rate constant variation over LPR5, LPR10, and LPR50 for the WT NeRh50 with NH4+ (figure 4c).

The Dataset

The data used for the figure are shown in Table 1

Show the code 
DT::datatable(dfms$dfm_5a, options=list(pageLength=5))
Table 1: Maximum amplitude and decay rate constant k for NH₄⁺ and MeA (figure 5a).

Downloads

The Models

A t-test would likely be fine here but, because a t-test is just a special case of a linear model, for consistency we again use linear models for amplitude and decay rate as dependent variables, with ligand as the independent factor, checking fit in Figure 1.

Show the code 
# Fit the models
model5a_amp_lm <- lm(amplitude ~ ligand, data=dfms$dfm_5a)
model5a_k_lm <- lm(decayrate ~ ligand, data=dfms$dfm_5a)

# Check model fit
performance::check_model(model5a_amp_lm)
performance::check_model(model5a_k_lm)
(a) amplitude ~ ligand
(b) decayrate ~ ligand
Figure 1: Diagnostic plots for linear model fits of amplitude ~ ligand and decayrate ~ ligand. The splines for linearity/heteroscadicity are odd, but the datapoints are not concerning. There is some evidence of non-normality of residuals in the maximum amplitude data, but nothing especially concerning.

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

Show the code 
EMM <- emmeans::emmeans(model5a_amp_lm, ~ ligand)
confint(emmeans::contrast(EMM, "pairwise", adjust="sidak")) |>
  kableExtra::kbl(digits=3) |>
  kableExtra::kable_styling(
    bootstrap_options = c("hover", "responsive")
  )
EMM <- emmeans::emmeans(model5a_k_lm, ~ ligand)
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. No contrast includes zero in the 95% confidence interval estimate.
(a) Maximum amplitude
contrast estimate SE df lower.CL upper.CL
NH4 - MeA 1.223 0.074 15 1.066 1.38
(b) Decay rate
contrast estimate SE df lower.CL upper.CL
NH4 - MeA 20.281 1.728 15 16.598 23.964

As no contrast includes zero in the 95% confidence interval, we conclude that there are differences in amplitude and decay rate constant for each pairwise comparison between ligands.

The Figures

We plot the differences, 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(model5a_amp_lm, ~ ligand),
                               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(model5a_k_lm, ~ ligand),
                               adjust="sidak")) |>
  tidyr::separate(contrast, into=c("group1", "group2"), sep=" - ") |>
  dplyr::mutate(y.position=85) |>
  rstatix::add_significance()

p5a_amp <- ggplot2::ggplot(dfms$dfm_5a, 
                           ggplot2::aes(x=ligand, y=amplitude, color=ligand)) +
  ggplot2::geom_boxplot(outlier.shape = NA) +
  ggplot2::geom_jitter() +
  ggplot2::labs(y="Maximum amplitude (nA)") +
  ggplot2::ylim(0, 3.5) +
  ggplot2::theme_minimal() +
  ggpubr::stat_pvalue_manual(EMM_amp,
                             label="p = {scales::pvalue(p.value)}",
                             size=3, step.increase=0.1)

p5a_k <- ggplot2::ggplot(dfms$dfm_5a, 
                         ggplot2::aes(x=ligand, y=decayrate, color=ligand)) +
  ggplot2::geom_boxplot(outlier.shape = NA) +
  ggplot2::geom_jitter() +
  ggplot2::labs(y="Decay rate constant k (/s)") +
  ggplot2::ylim(0, 105) +
  ggplot2::theme_minimal() +
  ggpubr::stat_pvalue_manual(EMM_k, 
                             label="p = {scales::pvalue(p.value)}", 
                             size=3, step.increase=0.1)

p5a_amp
p5a_k

# Write figures to file (commented out for webpage)
#ggplot2::ggsave("assets/images/fig5a_WT_maximum_amplitude.pdf", p5a_amp)
#ggplot2::ggsave("assets/images/fig5a_WT_maximum_amplitude.png", p5a_amp)
#ggplot2::ggsave("assets/images/fig5a_WT_decay_rate_constant.png", p5a_k)
#ggplot2::ggsave("assets/images/fig5a_WT_decay_rate_constant.pdf", p5a_k)
(a) Figure 5a, maximum amplitude
(b) Figure 5a, decay rate
Figure 2: WT measurements across two ligands, replicating figure 5a from the mansucript.

Downloads