Myosin Light Chain Kinase Controls Voltage-Dependent Calcium Channels in Vascular Smooth Muscle

myosin light chain kinase controls voltage-dependent calcium channels in vascular smooth muscle

A. Martinsen1, O. Schakman1, X. Yerna1, C. Dessy2 and N. Morel1.

(1) Cellular physiology, IoNS, UCLouvain, Brussels, Belgium

(2) Pharmacology and Therapeutics, IREC, UCLouvain, Brussels, Belgium

Corresponding author:

Pflügers Archiv - European Journal of Physiology.

Supplemental figure 1

Effect of voltage-dependent Ca2+ channels blockers on Ca2+ channel current in smooth muscle cells isolated from rat resistance mesenteric artery.

Current-voltage (I-V) curves showed that IBa started at -30 mV and reached a current peak at 10 mV. Current was unaffected by the P/Q channels blocker -agatoxin, but inhibited by the L-type Ca2+ channel blocker verapamil (10 µM) (70 ± 4 % (n=5) inhibition at 0 mV) and completely abolished when mibefradil (10 µM) was added to the perfusion solution in the presence of verapamil to further block T-type Ca2+ channel. Current was normalised to its value before perfusion with the blocker. Holding potential was maintained at -80 mV. Stimulation protocol as described under Methods.

Supplemental figure 2

MLCK downregulation and voltage-operated Ca2+ channels blockers inhibit Ca2+ release induced by thapsigargin and store-depletion induced Ca2+ entry in A7r5 cells.

a. Ca2+ response to thapsigargin was markedly attenuated in A7r5 cells transfected with MLCK-siRNA compared to cells transfected with a scramble siRNA.

Left panel: Time-course of changes in cytosolic Ca2+ in MLCK depleted or control A7r5 cells stimulated with thapsigargin (1 µM) in Ca2+-free medium and after addition of Ca2+ into the medium. Traces are mean values from 6 experiments. SEM are represented by vertical bars at representative time points.

Right panel: Change in cytosolic Ca2+ signal measured in the presence of thapsigargin in Ca2+ free medium (Ca2+ 0) and after the re-addition of Ca2+ in the perfusion solution in MLCK-siRNA (black filled bars) and scramble-siRNA (open bars) transfected A7r5 cells (n=6).

b. Ca2+ release and store-depletion induced Ca2+ entry were markedly depressed in the presence of verapamil and mibefradil.

Left panel: Time-course of changes in cytosolic Ca2+ in untransfected A7r5 control cells (light grey trace) or in cells incubated with verapamil plus mibefradil (10 µM, black trace) (n=5-6). SEM are represented by vertical bars at representative time points.

Right panel: Effect of voltage-dependent Ca2+ (VOC) channels blockers on Ca2+ signal in non-transfected A7r5 cells. Cytosolic Ca2+ signal was measured in the presence of thapsigargin (1 µM) in Ca2+-free solution (Ca2+ 0) and after the re-addition of Ca2+ in the perfusion solution in control cells (open bars) and in cells treated with verapamil plus mibefradil 10 µM (black filled bars) (n= 5-6).

*, **, *** indicate significant difference between scramble-siRNA-transfected or control cells and MLCK-siRNA-transfected or blockers-treated cells (* p<0.05, ** p<0.01, *** p<0.001).

Supplemental figure 3

Effect of MLCK depletion by a second MLCK-siRNA on Ca2+ signal and genes expression in A7r5 cells.

a. Cytosolic Ca2+ signal was recorded after AVP (10 nM) was applied in Ca2+-free medium (Ca 0) and after the re-addition of Ca2+ in the perfusion solution in the continuous presence of AVP (Ca AVP) in MLCK-siRNA 2 (black filled bars) and scramble-siRNA (open bars) transfected A7r5 cells (n=3). Results are expressed as mean ± SEM (vertical bars). * p<0.05 versus scramble-siRNA transfected cells. Ca2+ release induced by AVP in the absence of Ca2+ in the perfusion solution was significantly depressed in MLCK-depleted A7r5 cells. Ca2+ entry after the re-addition of Ca2+ in the perfusion solution in the presence of AVP was decreased in MLCK-siRNA 2 transfected A7r5 cells compared to scramble-siRNA transfected cells, although this decrease was not statistically significant (p=0.13). This result confirmed the result obtained with the other MLCK-siRNA used in the present paper.

b. Relative expression of voltage-operated Ca2+ channels, L-type (CaV1.2) and T-type (CaV3.1), in MLCK-siRNA 2 transfected A7r5 (black closed columns) compared to scramble-siRNA transfected cells (open columns) (n=4-6). Results are expressed as mean ± SEM (vertical bars). * p<0.05 and ** p<0.01 versus scramble-siRNA transfected cells. This result showed that MLCK gene expression was downregulated, although in a lower extent than in MLCK-siRNA transfected cells (fig. 6). The results also confirmed that MLCK depletion decreased the expression of voltage-operated Ca2+ channels, as demonstrated in the present paper (fig 6). As the expression of MLCK is less depressed by MLCK-siRNA 2 transfection than by MLCK-siRNA, we observed that the expression of CaV1.2 also was less affected.

1

Martinsen et al. Supplemental figures