Examining the neglected side of calcium regulation in taste cells.

Taste cells are excitable cells, heterogeneous in both form and in function. They detect stimuli by relaying information from external receptors to either hemichannels or classical synapses. As this process is governed by intracellular calcium, disruption of this delicate homeostasis would render the cell unresponsive and physiologically useless. The overwhelming majority of publications on calcium signalling concern intracellular calcium elevation, with seemingly little thought on how this calcium is subsequently removed from the cell. It must, however, be as tightly regulated a process as the original calcium elevation, for the refractory period to be sufficiently short for cell signaling to retain function. With this in mind, a recent article published in The Journal of Physiology by Laskowski & Medler (2009) represents one of the first attempts to explain calcium removal from taste cells. Their work hypothesizes that the superfamily of sodium–calcium exchangers (NCX) balances a constitutive calcium entry into taste cells, with varying efficacy related to taste cell subtype. In a generic excitable cell, at steady state, the same amount of calcium moving across the plasma membrane during calcium entry, be it from signaling events or from a constitutive entry, must be removed again across the plasma membrane. This would occur principally through the sodium–calcium exchanger, with the plasma membrane calcium ATPase (PMCA) playing a smaller role (see ‘1’ in Fig. 1). Calcium released from intracellular stores (the endoplasmic or sarcoplasmic reticulum) will be balanced principally by the SERCA or sarcoplamic/endoplasmic reticulum calcium ATPase pump (see ‘2’ in Fig. 1). In addition, a small contribution will be made to intracellular calcium buffering by the mitochondria. When at steady state, calcium removal, principally by the mitochondrial calcium uniporter (MCU) will be largely balanced by the Na+-dependent and Na+-independent mitochondrial calcium efflux mechanisms (see ‘3’ in Fig. 1). This paper represents the first to assay the relative contributions of each of these processes to intracellular calcium homeostasis as a whole. Figure 1 Intracellular calcium homeostasis in a generic exciteable cell is maintained via balance across the plasma membrane, mitochondria and the internal stores (endoplasmic/sarcoplasmic reticulum). Results It has been suggested previously (Vinnikova et al. 2003) that ionic exchangers are present in taste buds and the olfactory system (Reisert & Matthews, 1998). Laskowski and Medler confirm Na+–Ca2+ exchangers are present in taste buds in Fig. 6 of their work. The presence of mRNA for all subtypes of NCX and at least 4 out of 6 subtypes of the functionally similar sodium–calcium–potassium exchangers (NCKX) are demonstrated through RT-PCR, using brain as a control. This is confirmed functionally in their Fig. 1, when replacement of extracellular sodium with lithium renders the NCXs non-functional, resulting in an increase in intracellular calcium. Replacement of sodium with N−methyl-d-glucamine (NMG) instead of lithium yielded similar results, suggesting this was not an undefined effect due to the presence of lithium. Likewise, a pharmacological inhibition of the NCXs (their Fig. 3) yielded a similar outcome, suggesting the effect observed was not simply due to the absence of extracellular sodium. Pharmacological block of NCX reverse mode confirmed that the calcium increase was not merely due to NCX reversal. Not only does this suggest that NCXs have some contribution to the maintenance of intracellular calcium homeostasis, but that they remain functional at all times, not merely after a calcium signalling event, suggesting a constitutive calcium influx in taste cells. Their Fig. 4 pharmacologically identifies TRPV1, or a taste-specific TRPV1 variant, as the leak channel in question, using a blocker with a reportedly high degree of selectivity. In probably the most intriguing result in this work, Fig. 2 of Laskowski and Medler's work assays variation in calcium increase during NCX inhibition across taste cell types. Type III, or presynaptic taste cells showed significantly larger calcium increase when compared to type II, or receptor cells. Why this is so interesting is that presynaptic cells express voltage-gated calcium channels (VGCCs), and thus the increase in intracellular calcium in response to taste stimuli is from an extracellular source. This would result in the Ca2+ having to be removed across the plasma membrane, hence via the NCX molecules. Receptor cells do not possess these VGCCs; taste stimuli activate G-protein coupled receptors (GPCRs) within receptor cells, which couple to intracellular calcium stores. Calcium elevations in the case of receptor cells would be principally dealt with by the SERCA pump, and thus receptor cells might very well justify a lower expression level of NCXs. One possible concern regarding the methodology within this section would be that cell type was identified using 10 mm denatonium benzoate. This would succeed in identifying only denatonium-sensitive cells, not receptor cells. In addition to this, denatonium at this high concentration can stimulate calcium responses in non-type II cells (Vandenbeuch et al. 2008) in addition to type II responses. Indeed, in the previous publication from this group (Hacker et al. 2008), they highlight that this method of identifying cell types resulted in a small population of taste cells which responded both to denatonium and KCl. Aside from this, however, their demonstration that presynaptic cells show a higher contribution to calcium homeostasis from NCXs fits in very nicely with established literature and the reader's own intuitive views on calcium homeostasis across taste cell types. Their Fig. 5 uses a protonophore, carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP), to inhibit mitochondrial buffering of intracellular calcium, leading to the conclusion that mitochondria contribute to the buffering of a constitutive calcium entry in taste cells. This could, however, only be the case in the short term. Once the intracellular calcium level in taste cells reaches equilibrium, a constitutive calcium entry from TRPV1 or TRPV1-like channels could only be balanced by calcium removal from the cell, most probably through either NCX or plasma membrane calcium ATPase (PMCA) pumps. For mitochondria to continuously buffer a constitutive calcium entry at steady state would cause mitochondrial calcium to elevate infinitely, a situation which cannot occur. An alternative interpretation which I would like to offer to the data within this figure is that as the cyanide-derived protonophore FCCP operates through the inhibition of ATP synthesis, a dramatic decrease in intracellular ATP is inevitable with its use (Tretter et al. 1998). A lack of intracellular ATP would lead to a number of physiological alterations in calcium homeostasis within the cell, including, but not limited to, slowing of SERCA pumps, PMCAs and Na–K ATPases. The slowing of SERCA pumps would only inhibit the refilling of intracellular calcium stores, and hence not the balance concerned with a constitutive calcium entry into the cells. However, inhibiting PMCAs, or indeed Na–K ATPases, which are concerned with the removal of sodium from the cytosol, and therefore are critical to NCX function, would cause such a calcium increase. This may also account for the subsequent non-additive relationship observed between lithium-dependent, FCCP-dependent, and lithium + FCCP-dependent calcium increase.