Differential targeting and signalling of voltage‐gated T‐type Ca_v3.2 and L‐type Ca_v1.2 channels to ryanodine receptors in mesenteric arteries

Abstract

Recent data suggest that T-type Ca 3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca signalling (Ca sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Ca 3.2 channels and their functional interaction with Ca 1.2 channels to trigger Ca sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen-inducible smooth muscle-specific Ca 1.2 (SMAKO) mice and laser scanning confocal microscopy to assess Ca spark generation in VSMCs. Ni , Cd and methyl-β-cyclodextrin were used to inhibit Ca 3.2 channels, Ca 1.2 channels and caveolae, respectively. Ni (50 μmol L ) and methyl-β-cyclodextrin (10 mmol L ) decreased Ca spark frequency by ∼20-30% in mesenteric VSMCs in a non-additive manner, but failed to inhibit Ca sparks in tibial and cerebral artery VSMCs. Cd (200 μmol L ) suppressed Ca sparks in mesenteric arteries by ∼70-80%. A similar suppression of Ca sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca sparks were fully abolished by Ni or methyl-β-cyclodextrin. Our data demonstrate that Ca influx through Ca 1.2 channels is the primary means of triggering Ca sparks in murine arterial VSMCs. Ca 3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca source for Ca spark generation in mesenteric, but not tibial and cerebral, arteries.

Age attenuates the T‐type CaV3.2‐RyR axis in vascular smooth muscle

“…The T‐type Ca v 3.2 channel blocker Ni 2+ decreased Ca 2+ spark frequency and fraction of cells with sparks in young VSMCs (see also (Fan et al, 2018; Hashad et al, 2018)), while it failed to decrease Ca 2+ spark events in old VSMCs (Figure 1). These data suggest that Ca v 3.2 channels contribute to generation of Ca 2+ sparks in young but not in old VSMC.…”

“…The data also show that SR Ca 2+ load is controlled by SERCA (Nelson et al, 1995). We next studied how Ca v 1.2 channel ablation and reduced [Ca 2+ ] SR load affect the Ca V 3.2‐RyR axis, that is, direct coupling between Ca V 3.2 channels and RyRs to generate Ca 2+ sparks (Fan et al, 2018; Hashad et al, 2018; Löhn et al, 2000). Consistent with our previous results (Essin et al, 2007), we found that [Ca 2+ ] SR was lower in Ca v 1.2 −/− (SMAKO) VSMCs compared to Ca v 1.2 +/+ control cells.…”

“…STOCs were measured in presence of Cd 2+ and/or Ni 2+ after depletion of the [Ca 2+ ] SR by thapsigargin. The holding potential was set to −40 mV, a physiological membrane potential that should drive T‐type Ca 2+ channel‐mediated Ca 2+ sparks, enabling the activation of BK Ca channels (Fan et al, 2018; Harraz et al, 2014; Hashad et al, 2018). Figure shows that thapsigargin removed ~60% of STOCs in VSMCs (Figure e–g).…”

“…As a result, Ca 2+ spark–BK Ca channel coupling induces vascular smooth muscle cell (VSMCs) hyperpolarization and attenuation of arterial constriction (Brenner et al, 2000; Löhn et al, 2001; Pérez, Bonev, Patlak, & Nelson, 1999). In previous studies, we demonstrated that L‐type Ca V 1.2 channels play the predominant role (~75%) in Ca 2+ sparks generation in mesenteric arterial VSMCs, and T‐type Ca V 3.2 channels, localized in caveolae, represent an additional source (~25%) (Fan, Kaßmann, Hashad, Welsh, & Gollasch, 2018; Hashad et al, 2018). In the latter pathway, caveolae position Ca V 3.2 channels sufficiently close to RyRs (<40 nm) of the sarcoplasmic reticulum (SR) for extracellular Ca 2+ influx to trigger Ca 2+ sparks and large‐conductance Ca 2+ ‐activated K + channel feedback in vascular smooth muscle (Fan et al, 2018; Harraz et al, 2014; Hashad et al, 2018; Löhn et al, 2000).…”

“…In previous studies, we demonstrated that L‐type Ca V 1.2 channels play the predominant role (~75%) in Ca 2+ sparks generation in mesenteric arterial VSMCs, and T‐type Ca V 3.2 channels, localized in caveolae, represent an additional source (~25%) (Fan, Kaßmann, Hashad, Welsh, & Gollasch, 2018; Hashad et al, 2018). In the latter pathway, caveolae position Ca V 3.2 channels sufficiently close to RyRs (<40 nm) of the sarcoplasmic reticulum (SR) for extracellular Ca 2+ influx to trigger Ca 2+ sparks and large‐conductance Ca 2+ ‐activated K + channel feedback in vascular smooth muscle (Fan et al, 2018; Harraz et al, 2014; Hashad et al, 2018; Löhn et al, 2000). These conclusions were mainly derived from experiments using Ca V 3.2 channel ( Cacna1h −/− ) and caveolin‐1 ( Cav1 −/− ) knockout mice.…”