Direct block of Ca2+ channels by calmidazolium in cultured vascular smooth muscle cells.
We investigated the action of calmidazolium (CMZ), an inhibitor of calmodulin (CaM), on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), by using the whole-cell voltage-clamp method. All experiments were conducted at room temperature (24-25 degrees C). The peak IBa (Ca2+ channel current with 5 mM Ba2+ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of -60 mV. To elevate intracellular free Ca2+ concentration ([Ca]i) to pCa 6.5, the pipette solution contained a Ca2+-EGTA buffer (pCa 6.5) to allow equilibration with the cells. Bath application of 1 microM CMZ reduced the peak amplitude of IBa to 36.7+/-4.9% (n = 8); maximal effect occurred within 7-8 min. Peak IBa continued to decrease even after washing out the CMZ. Recovery of IBa was not observed even after 10 min of washout. Even in presence of an peptide inhibitor of CaM-dependent protein kinase-II (5.2 microM) in the pipette solution, CMZ inhibited IBa to 27.8 +/-5.3% (n = 7). To exclude the possibility that other Ca2+/ CaM-dependent kinases and phosphatases may regulate Ca2+ channel activity, we examined the effect of CMZ on IBa when [Ca]i was reduced by use of Ca2+/EGTA-buffered pipette solutions. At pCa approximately equal to 10 (10 mM EGTA and only contaminant Ca2+), CMZ inhibited IBa to 33.4+/-5.9% (n = 14) with a median inhibitory concentration (IC50) value of 0.29 microM. The activation curve (pCa approximately equal to 10) was shifted in the positive direction by 6.3 mV; the inactivation curve was shifted in the negative direction by 5.0 mV. CMZ decreased IBa progressively during repetitive step depolarizations. CMZ did not slow the rate of recovery from inactivation. In conclusion, CMZ inhibits Ca2+ channel current in a use-dependent manner. This inhibition is independent of CaMK-II and other Ca2+/CaM-dependent pathways. Therefore it is likely due to direct blockade of Ca2+ channels by CMZ. CMZ may reduce the outer surface charge and block the open state of the Ca2+ channels.
Sunagawa
M
M
Department of Molecular and Cellular Physiology, College of Medicine, University of Cincinnati, Ohio 45267-0576, USA. sunagam@email.uc.edu
Yokoshiki
H
H
Seki
T
T
Nakamura
M
M
Laber
P
P
Sperelakis
N
N
D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects*; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D018408:Patch-Clamp Techniques; D047428:Protein Kinase Inhibitors; D013997:Time Factors
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; C031938:calmidazolium; D004533:Egtazic Acid