Description

Calcium homeostasis is essential for normal body function. Calx is a Drosophila homolog of the mammalian sodium/calcium exchanger (NCX) involved in the regulation of intracellular calcium (Ca²⁺) level (Schwarz and Benzer 1997). A major function of Calx is to export excess Ca²⁺ to the outside of the cell when intracellular Ca²⁺ level is elevated (Hryshko et al. 1996). Genetic studies in Drosophila have shown that Calx mutants can develop to adult flies, even though Calx is expressed throughout the developmental stages (Wang et al. 2005). However, Ca²⁺ efflux mediated by Calx is crucial for maintaining the cellular Ca²⁺ homeostasis in light-activated sensory neurons (Wu et al. 2011). In addition to its role for phototransduction, Calx is also required for preventing light-induced retinal degeneration caused by Ca²⁺ overload (Wang et al. 2005).

Thus far, Calx function has been extensively studied in the retinal cells. However, expression of Calx gene is not restricted to the eye, and potential functions of Calx in other tissues are largely unknown. Interestingly, disrupted Ca²⁺ homeostasis has been implicated in aging and is regarded as one of the biomarkers of aging (Foster and Kumar 2002). Although mammalian NCXs have been implicated in aging of tissues and organs (Gomez-Villafuertes et al. 2007; Zhang et al. 2014), it remains to be studied whether NCXs are important for animal lifespan. Here, we address the question whether Calx affects lifespan in fly.

To determine whether Calx is required for normal lifespan, we carried out genetic analysis using two Calx mutants, Calx^A and Calx^B. It has been shown that homozygous Calx^A and Calx^B mutant flies are viable (Wang et al. 2005). Calx^A mutants have a point mutation (T822I) but show relatively normal level of protein expression in adult head. In contrast, Calx^B is a regulatory mutation that causes reduced protein expression (Wang et al. 2005). To check whether such mutant effects can also be seen in non-head tissues, we examined Calx protein expression in whole adult fly body. It has been reported that Calx is expressed as a single 110 kD protein in adult head and body (Wang et al., 2005). w¹¹¹⁸ flies used as wild-type control showed a major band at 110 kD and a weaker band at 120 kD (Fig. 1A). Consistent with earlier studies (Wang et al. 2005), we found a strong reduction in the 110 kD band in Calx^B mutant, while Calx^A mutant showed a nearly indistinguishable pattern from wild-type. Calx^B mutant showed severe reduction in both 110 and 120 kD bands, suggesting that Calx exists at least in two different forms in adult fly.

Next, we measured lifespan of wild-type and Calx males. We used isogenic w¹¹¹⁸ as control since it was previously used as control for analyzing Calx mutant phenotypes in phototransduction (Wang et al. 2005). In our lifespan assay at 25 ^oC, both Calx^A and Calx^B mutants showed considerable reduction in the survival rate compared with the wild-type control (Fig. 1B). Median lifespan of Calx^A and Calx^B mutants was 24 and 21.5 days, respectively, whereas the median for wild-type was about 36 days (Fig. 1D). We also examined survival rates of Calx mutants at 29 ^oC (Fig. 1C). At this high temperature, wild-type flies showed a lower survival rate with median lifespan of 21.5 days. Under this condition, Calx^A mutant flies had a significantly shorter median lifespan than Calx^B mutant (12.5 days for Calx^A; 17.7 days for Calx^B) (Fig. 1D). Since Calx^A encodes a mutated protein, it is possible that the mutant Calx protein might be more unstable at 29 ^oC, causing more severe phenotype in longevity. Alternatively, Calx^A mutant cells may require a higher rate of calcium extrusion at the upper extreme of their viable temperature range than their more normal growth temperature (25 ^oC). We also checked the lifespan of transheterozygotes for Calx^A and Calx^B mutations (Calx^A/Calx^B). Trans-heterozygous flies also showed significantly reduced lifespan compared with wild-type, indicating that Calx^A and Calx^B fail to complement the longevity phenotype. Consistent with the hypomorphic nature of the Calx^B allele, trans-heterozygous flies showed an intermediate lifespan between Calx^A and Calx^B mutants (Fig. 1C, D). Taken together, our data suggest that the Calx mutations are responsible for the observed phenotype of shorter longevity. These results are preliminary, and to rule out potential genetic background effects, additional controls should be included in the future.

This study raises a possibility that Calx might be required for normal lifespan. The Ca²⁺ influx channel TRP, which has an opposite function to Calx, is crucial for phototransduction (Montell 2005; Wang et al. 2005). The trp gene has a paralog, trp-like (trpl) (Phillips et al. 1992). Although there is no strict paralog of Calx in Drosophila, Nckx30C is a distant relative of Calx (Haug-Collet et al. 1999). It remains to be tested whether these related genes are also required for normal lifespan. Importantly, retinal degeneration by loss of TRP or constitutive TRP function can be suppressed by defects or overexpression of Calx, respectively. Further studies are necessary to see whether lifespan is dependent on the functional relationship between Calx and TRP in the intracellular Ca²⁺ regulation, although such genetic analysis may be complicated by the existence of related genes like trpl and Nckx30C. It is also an intriguing question whether NCX homologs of Calx play a role in lifespan of mammals.