Analysis of Conditional Paralytic Mutants in Drosophila Sarco-Endoplasmic Reticulum Calcium ATPase Reveals Novel Mechanisms for Regulating Membrane Excitability

Abstract

Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function. At motor nerve terminals, SERCA inhibition retards calcium sequestration and reduces the amplitude of evoked excitatory junctional currents. This suggests a direct contribution of store-derived calcium in determining the quantal content of evoked release. Conditional paralysis of SERCA mutants is also marked by prolonged neural activity-driven muscle contraction, thus reflecting the phylogenetically conserved role of SERCA in terminating contraction. Further analysis of ionic currents from mutants uncovers SERCA-dependent mechanisms regulating voltage-gated calcium channels and calcium-activated potassium channels that together control muscle excitability. Finally, our identification of dominant lossof-function mutations in SERCA indicates novel intra-and intermolecular interactions for SERCA in vivo, overlooked by current structural models.

The effects of ER morphology on synaptic structure and function in Drosophila melanogaster

“…ER-localized Ca 2+ release channels such as the inositol 1,4,5-trisphosphate (IP3) receptor, the ryanodine receptor and the TRPV1 channel play key roles in evoked neurotransmitter release (Emptage et al, 2001;Liang et al, 2002;Llano et al, 2000;Wong et al, 2014). In addition, dominantnegative mutations in the Drosophila ER-localized Ca 2+ pump SERCA decrease evoked transmitter release by ∼50% (Sanyal et al, 2005), which is consistent with the possibility that ER-derived Ca 2+ contributes significantly to the Ca 2+ required to trigger transmitter release.…”

Synaptic neuropeptide release induced by octopamine without Ca 2+ entry into the nerve terminal

“…Releasing presynaptic endoplasmic reticulum (ER) Ca 2+ with caffeine (21) for 3 min elicited release of 24 ± 4% of synaptic neuropeptide content, suggesting a possible role for ER Ca 2+ in presynaptic octopamine action. Therefore, to genetically probe whether ER Ca 2+ stores participated in the octopamine effect, neuropeptide release was studied in the CaP60A Kum170 (Kum170) mutant, in which the sarco-ER Ca 2+ ATPase (SERCA) is persistently inhibited by brief exposure to 40°C (22). Although octopamine evoked release under permissive conditions, heating Kum170 animals for 8 min to deplete ER Ca 2+ stores abolished release measured Release induced by the epac activator (WT+Me, n = 5) was abolished in epac mutant flies (mut+Me, n = 5), but FSK-evoked release in WT animals (WT+FSK, n = 8) was still apparent in the epac mutant (mut+FSK, n = 3).…”

Peptide-Induced Modulation of Synaptic Transmission and Escape Response inDrosophilaRequires Two G-Protein-Coupled Receptors

“…The CaP60A Kum170 mutants, which fail to pump calcium back into the endoplasmic reticulum (ER) at high temperatures, were used to deplete calcium stores in the ER. Calcium ATPase function on the endoplasmic reticulum can be disrupted in these mutants by incubating them in Ca 2ϩ -free HL3 at 40°C for 8 min, preventing the refilling of the calcium stores (Sanyal et al, 2005 ; P{TRiP.JF01850}attP2) using either a muscle-specific (24b-GAL4 ) or neuron-specific driver (elav-GAL4 ).…”