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A fine‐tuned interaction between the ER and mitochondria regulates Ca 2+ ‐dependent after hyperpolarizations in vasopressin supraoptic neurons
Author(s) -
Kirchner Matthew Karl,
Stern Javier Eduardo
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05458
Subject(s) - serca , thapsigargin , endoplasmic reticulum , neuroscience , chemistry , microbiology and biotechnology , gating , biophysics , biology , biochemistry , atpase , enzyme
It is known that vasopressin magnocellular neurons become hyperexcitable in cardiovascular diseases such as heart failure. This hyperexcitability is mediated by shifts in both synaptic and intrinsic mechanisms. One key intrinsic mechanism is the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium‐dependent K + current ( I sAHP ) most notable for its role in spike frequency adaptation. While features of the sAHP and its effects on firing are well established, a comprehensive understanding of the underlying mechanisms are still unknown. Previous work demonstrated that sAHP activation requires Ca 2+ entry via voltage‐gated Ca 2+ channels (VGCCs). However, whether in VP neurons Ca 2+ entry via these channels directly activates the sAHP, or alternatively, whether it stimulates Ca 2+ ‐induced Ca 2+ release from an internal source such as endoplasmic reticulum (ER) or mitochondria remains unknown. Combining patch clamp electrophysiology with simultaneous Ca 2+ imaging in transgenic eGFP‐VP rats, we investigated the role of ER and mitochondrial in shaping the sAHP. We measured sAHPs in voltage clamp by evoking a train of action potentials and measuring the resulting outward current. Pre‐incubation of slices with thapsigargin (3μM), an inhibitor of the sarco/ER Ca 2+ ATPase inhibited I sAHPs significantly (96%, p< 0.001) while leaving Ca 2+ transients unaffected (p > 0.05). This demonstrates that ER Ca 2+ release is necessary for sAHP activation. Conversely, blockade of mitochondrial Ca 2+ release via blocking mitochondrial Ca 2+ exchangers with TPP (20μM) failed to inhibit the I sAHP (p > 0.05). We next evaluated the role of mitochondrial Ca 2+ uptake with bath application of the mitochondrial uncoupler CCCP (1μM) or the mitochondrial Ca 2+ uniporter (MCU) blocker Ru360 (20μM) in the patch pipette. These manipulations significantly prolonged the decay time constant of both the I sAHP (p< 0.01) and the underlying Ca 2+ transient (p< 0.05), demonstrating that the MCU regulates spatiotemporal dynamics of the sAHP. Finally, combining thapsigargin with Ru360 in the pipette showed an initial block of the sAHP, followed by an enhancement over 15 minutes as Ru360 dialyzed into the cell. These results support the ER (but not mitochondria) as a major source of Ca 2+ activating the sAHP, as well as a role for the MCU in regulating the magnitude and time course of the sAHP. Taken together, our studies support a fine‐tuned interaction between VGCCs, ER, and mitochondria in modulating the magnitude and time course of the sAHP in VP neurons. Future studies warrant investigation into whether changes in these interactions contribute to altered VP neuronal activity in cardiovascular disease states. Support or Funding Information R01HL090948