Differential targeting and signalling of voltage-gated T-type Ca_v 3.2 and L-type Ca_v 1.2 channels to ryanodine receptors in mesenteric arteries

Key points: In arterial smooth muscle, Ca²⁺ sparks are elementary Ca²⁺ -release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca²⁺ -sensitive K⁺ (BK_Ca ) channels. This study elucidated the contribution of T-type Ca_v 3.2 channels in caveolae and their functional interaction with L-type Ca_v 1.2 channels to trigger Ca²⁺ sparks in vascular smooth muscle cells (VSMCs). Our data demonstrate that L-type Ca_v 1.2 channels provide the predominant Ca²⁺ pathway for the generation of Ca²⁺ sparks in murine arterial VSMCs. T-type Ca_v 3.2 channels represent an additional source for generation of VSMC Ca²⁺ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T-type Ca_v 3.2 channels and RyRs to ignite Ca²⁺ sparks.

Abstract: Recent data suggest that T-type Ca_v 3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca²⁺ signalling (Ca²⁺ sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca²⁺ sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Ca_v 3.2 channels and their functional interaction with Ca_v 1.2 channels to trigger Ca²⁺ sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen-inducible smooth muscle-specific Ca_v 1.2^-/- (SMAKO) mice and laser scanning confocal microscopy to assess Ca²⁺ spark generation in VSMCs. Ni²⁺ , Cd²⁺ and methyl-β-cyclodextrin were used to inhibit Ca_v 3.2 channels, Ca_v 1.2 channels and caveolae, respectively. Ni²⁺ (50 μmol L^-1 ) and methyl-β-cyclodextrin (10 mmol L^-1 ) decreased Ca²⁺ spark frequency by ∼20-30% in mesenteric VSMCs in a non-additive manner, but failed to inhibit Ca²⁺ sparks in tibial and cerebral artery VSMCs. Cd²⁺ (200 μmol L^-1 ) suppressed Ca²⁺ sparks in mesenteric arteries by ∼70-80%. A similar suppression of Ca²⁺ sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca²⁺ sparks were fully abolished by Ni²⁺ or methyl-β-cyclodextrin. Our data demonstrate that Ca²⁺ influx through Ca_V 1.2 channels is the primary means of triggering Ca²⁺ sparks in murine arterial VSMCs. Ca_V 3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca²⁺ source for Ca²⁺ spark generation in mesenteric, but not tibial and cerebral, arteries.