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μ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants
Mark Hnatowich1, Hoa Dinh Le, Danielle DeMoissac
1Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, University of Manitoba, Faculty of Medicine, 351 Tache Avenue, Winnipeg, Manitoba, Canada.
Abstract:
μ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of μ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to μ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) μ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) μ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after μ-calpain treatment. The ability of μ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.
Insights
The protease μ-calpain disrupts calcium (Ca2+)-dependent regulation of sodium-calcium exchangers (NCX1), leading to constitutive activation of exchange currents. This finding has implications for tissues expressing these NCX1 variants.
Area of Science:
- Biochemistry
- Molecular Biology
- Physiology
Background:
- μ-Calpain is a calcium-activated protease found in mammalian tissues.
- Sodium-calcium exchangers (NCX1) play crucial roles in regulating intracellular calcium levels.
- NCX1 exists in alternatively spliced variants (NCX1.1, NCX1.3, NCX1.4) with distinct regulatory properties.
Purpose of the Study:
- To investigate the impact of μ-calpain on the functional regulation of three NCX1 splice variants.
- To determine how μ-calpain affects Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulation of NCX1.
- To explore the potential pathophysiological consequences of μ-calpain's action on NCX1.
Main Methods:
- Utilized the giant, excised patch technique in Xenopus oocytes.
- Expressed specific NCX1 splice variants (NCX1.1, NCX1.3, NCX1.4) in oocytes.
- Assessed the regulatory phenotypes (I(1) inactivation and I(2) regulation) of NCX1 before and after μ-calpain treatment.
Main Results:
- μ-Calpain selectively abolished Ca(2+)-dependent (I(2)) regulation, causing constitutive activation of NCX1 currents.
- μ-Calpain had minimal effects on Na(+)-dependent (I(1)) regulation, causing only slight destabilization.
- Higher intracellular Ca(2+) levels alleviated I(1) inactivation in NCX1.1 and NCX1.4, a mechanism largely unaffected by μ-calpain.
Conclusions:
- μ-Calpain treatment leads to constitutive activation of Na(+)-Ca(2+) exchange currents by disrupting I(2) regulation.
- Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) inactivation are distinct mechanisms.
- The selective activation of NCX1 by μ-calpain may have significant pathophysiological implications in relevant tissues.
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