Activation of Na+/H+ and K+/H+ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flu

Citation:

Alejandro Ortiz-Acevedo, Robert R. Rigor, Hector M. Maldonado, and Peter M. Cala

Activation of Na⁺/H⁺ and K⁺/H⁺ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity
Am J Physiol Cell Physiol 295: C1316-C1325, 2008. First published doi:10.1152/ajpcell.00160.2008

Summary:

In this paper, the authors sought to study the mechanism behind the Na⁺/H⁺ and K⁺/H⁺ exchange that is crucial to cell volume maintenance. Cells regulate their volume when placed in hypo- or hyper- osmotic environments through the efflux of K⁺ and Cl^- (when swelled) and the influx of Na⁺ and Cl^- (when shrunk). These channels have been shown to be affected by phosphorylation and dephosphorylation cascades, but the mechanism is not understood. Based on previous studies, the authors hypothesize a phosphorylation-based scheme for the regulation of intercellular volume, and test this mechanism through a variety of conditions. These experiments are based on the inhibitory properties of calyculin A (CLA), ouabain, and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). CLA is a phosphotase inhibitor, so introduction into culture will result in up-regulation of phosphorylation cascades. Ouabain is a Na⁺/K⁺ ATPase inhibitor, and is used to eliminate alternative sources of ion flux. EIPA is an inhibitor of both the Na⁺/H⁺ antiport and potentially of the K⁺/H⁺ antiport. Together, these three molecules are used to regulate ion flux into and out of a cell during osmotic stresses.

The effect of CLA on cells in isotonic solutions was measured by measuring the flux of radio-labeled ions in solution. Even without osmotic stresses, cells exposed to CLA demonstrated a net Na⁺ uptake and a net K⁺ loss (Figure 1). This is interesting, because upregulating phosphorylation will induce cell responses to both swelling and shrinking simultaneously. This suggests that both pathways are either controlled by a common regulatory event or sequentially linked in some way. To test the latter scenario, normal K⁺/H⁺ and Na⁺/H⁺ channel function was blocked by using ‘null media’ (media designed to maintain those channels at a thermodynamic valley to eliminate driving force for ion flow). When cells in this media were exposed to CLA, they continued to exhibit ion flux, which suggests that this flow is not secondary to another channel. Next, EIPA, a Na⁺/H⁺ Exchanger 1 (NHE1) inhibitor, was used to block access to Na⁺/H⁺ exchangers in the cell membrane, while also blocking homologous NHE’s that mediate K⁺/H⁺ exchange as well. Blocking of ion channels resulted in significantly decreased ion flux activity, even when cells were exposed to CLA (Figure 6). This further suggests that the mechanism for cell volume control runs through phosphorylation of K⁺/H⁺ and Na⁺/H⁺ channels.

The authors next seek to determine if both the cell swelling and cell shrinking responses are controlled by the same pathway. They propose that a shrinkage-activated kinase activates Na⁺/H⁺ exchange but is suppressed by swollen cells, and vice versa. To test this, a new experimental design was created to zero out phosphotase activity prior to exposure to osmotic stresses. The ‘null media’ was used again to ensure that no ion flow occurred while cells were incubated with CLA. After this incubation, cells are exposed to media without CLA and ion flow is measured. When an isotonic solution was used for this experiment, cells experienced no net ion flow during the incubation period, and a subsequent net influx of Na⁺ and efflux of K⁺ (Figure 7). However, when cells were incubated in a hypotonic solution, they experienced primarily a K⁺ efflux after exposure to non-‘null media’. Likewise, cells in a hypertonic solution experienced primarily a Na⁺ influx after media change (Figure 8). This result suggests that the pathways for each ion channel flux are independent of the other, but can be regulated at a common step by a CLA-sensitive phosphotase.

Significance:

This paper proposes and tests a novel mechanism for the control of cell volume through sodium and potassium channels. While prior work had already hinted at the link between phosphorylation and regulation of ion channels, this paper creates a model that explains selective ion flow through two independent pathways (one for cell swelling, one for cell shrinking) that share a common regulator step (for isotonic solutions).

Critique:

There were some potential systemic issues with their protocol that might adversely affect data validity. Throughout the experiments, rabbit red blood cells were held at 23ºC, which is significantly lower than body temperature (37ºC). The effect the lowered temperature may have on membrane permeability is unknown, and may skew results unpredictably. All ion flow measurements were taken using radioisotopes of ions and a gamma counter or beta scintillation counter. The authors described how they would cut the tube “just above the cell pellet to minimize contamination by extracellular isotope” during readings. However, this seems very inconsistent and irreproducible, since with varying sized pellets would come varying sized cuts, and the background radiation would be impossible to blank properly.

Additionally, I was not entirely convinced by their model for how the two pathways are related, while at the same time controlled upstream by a shared phosphotase. While their proposal seems entirely reasonable, the data do not conclusively point to this mechanism as the best or only one. While the experiments conducted have shown that the proposed mechanism is possible, I feel that further tests would need to be conducted to rule out alternative mechanisms. One particular area of interest is in the possibility that alternative ion channels played a part in this mechanism. While the authors tried to control for channels such as the Na⁺/K⁺ ATPase and the Na⁺/H⁺ or K⁺/H⁺ exchangers, it seems likely that there were additional ion channels that should have been accounted for. Overall, while this paper provides a lot of evidence supporting the author’s proposed mechanism, it does not provide evidence sufficiently disproving the possibility of alternative mechanisms.

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