Figure 6b: WT NeRh50 maximum amplitudes and decay rate constants after a 200 mM ammonium pulse in proteoliposomes containing NeRh50 at LPR10, at four pH conditions

Manuscript figure 6b shows WT maximum amplitude and decay rate constant variation after a 200 mM ammonium pulse in proteoliposomes containing NeRh50 at LPR10, at four pH conditions (figure 6b)

The Dataset

The data used for the figure are shown in Table 1

Show the code 
DT::datatable(dfms$dfm_6b, options=list(pageLength=5))
Table 1: Maximum amplitude and decay rate constant k for transient current measured after a 200 mM ammonium pulse in proteoliposomes containing NeRh50 at LPR10, at four pH conditions (figure 6b).

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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
model6b_amp_lm <- lm(amplitude ~ group, data=dfms$dfm_6b)
model6b_k_lm <- lm(decayrate ~ group, data=dfms$dfm_6b)

# Check model fit
performance::check_model(model6b_amp_lm)
performance::check_model(model6b_k_lm)
(a) amplitude ~ group
(b) decayrate ~ group
Figure 1: Diagnostic plots for linear model fits of amplitude ~ group and decayrate ~ group. The heteroscadicity spline is odd, but the datapoints are not particularly concerning. There is some evidence of non-normality of residuals in the decay rate constant data, but nothing especially concerning.

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

Show the code 
EMM <- emmeans::emmeans(model6b_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(model6b_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. All contrasts include zero in the 95% confidence interval estimate.
(a) Maximum amplitude
contrast estimate SE df lower.CL upper.CL
pH7 (baseline) - pH7 after pH5 0.053 0.169 13 -0.471 0.578
pH7 (baseline) - pH7 after pH6 -0.006 0.196 13 -0.612 0.600
pH7 (baseline) - pH7 after pH8 0.097 0.180 13 -0.460 0.653
pH7 after pH5 - pH7 after pH6 -0.060 0.196 13 -0.666 0.546
pH7 after pH5 - pH7 after pH8 0.043 0.180 13 -0.513 0.600
pH7 after pH6 - pH7 after pH8 0.103 0.205 13 -0.531 0.737
(b) Decay rate
contrast estimate SE df lower.CL upper.CL
pH7 (baseline) - pH7 after pH5 -0.25 2.847 13 -9.065 8.565
pH7 (baseline) - pH7 after pH6 -2.17 3.288 13 -12.348 8.008
pH7 (baseline) - pH7 after pH8 7.78 3.020 13 -1.569 17.129
pH7 after pH5 - pH7 after pH6 -1.92 3.288 13 -12.098 8.258
pH7 after pH5 - pH7 after pH8 8.03 3.020 13 -1.319 17.379
pH7 after pH6 - pH7 after pH8 9.95 3.438 13 -0.695 20.595

All contrasts include zero in the 95% confidence interval, so we conclude that there are no differences in amplitude and decay rate constant for each pairwise comparison between groups (pH conditions).

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(model6b_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(model6b_k_lm, ~ group),
                               adjust="sidak")) |>
  tidyr::separate(contrast, into=c("group1", "group2"), sep=" - ") |>
  dplyr::mutate(y.position=70) |>
  rstatix::add_significance()

p6b_amp <- ggplot2::ggplot(dfms$dfm_6b, 
                           ggplot2::aes(x=group, y=amplitude, color=group)) +
  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)

p6b_k <- ggplot2::ggplot(dfms$dfm_6b, 
                         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)") +
  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)

p6b_amp
p6b_k

# Write figures to file (commented out for webpage)
#ggplot2::ggsave("assets/images/fig6b_WT_maximum_amplitude.pdf", p6b_amp)
#ggplot2::ggsave("assets/images/fig6b_WT_maximum_amplitude.png", p6b_amp)
#ggplot2::ggsave("assets/images/fig6b_WT_decay_rate_constant.png", p6b_k)
#ggplot2::ggsave("assets/images/fig6b_WT_decay_rate_constant.pdf", p6b_k)
(a) Figure 6b, maximum amplitude
(b) Figure 6b, decay rate
Figure 2: Transient current maximum amplitude and decay rate constant measured after a 200 mM ammonium pulse in proteoliposomes containing NeRh50 at LPR10, for four pH regimes.

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