Direct block of Ca2+ channels by calmidazolium in cultured vascular smoothmuscle cells

We investigated the action of calmidazolium (CMZ), an inhibitor of calmodul in (CIM), on the L-type Ca2+ currents (I-Ca(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 I-Ba (Ca2+ channel current with 5 mM Ba2+ as charge carrier) w as evoked every 15 a by a test potential to +10 mV from a holding potential of -60 mV. To elevate intracellular free Ca2+ concentration ([Ca](i)) to p Ca 6.5, the pipette solution contained a Ca2+-EGTA buffer (pCa 6.5) to allo w equilibration with the cells. Bath application of 1 mu M CMZ reduced the peak amplitude of I-Ba to 36.7 +/- 4.9% (n = 8); maximal effect occurred wi thin 7-8 min. Peak I-Ba continued to decrease even after washing out the CM Z. Recovery of I-Ba was not observed even after 10 min of washout. Even in presence of an peptide inhibitor of CaM-dependent protein kinase-II (5.2 mu M) in the pipette solution, CMZ inhibited I-Ba to 27.8 +/- 5.3% (n = 7). T o exclude the possibility that other Ca2+/CaM-dependent kinases and phospha tases may regulate Ca2+ channel activity, we examined the effect of CMZ on I-Ba when [Ca](i) was reduced by use of Ca2+/EGTA-buffered pipette solution s. At pCa approximate to 10 (10 mM EGTA and only contaminant Ca2+), CMZ inh ibited I-ba to 33.4 +/- 5.9% (n = 14) with a median inhibitory concentratio n (IC50) value of 0.29 mu M. The activation curve (pCa = 10) was shifted in the positive direction by 6.3 mV; the inactivation curve was shifted in th e negative direction by 5.0 mV. CMZ decreased I-Ba progressively during rep etitive step depolarizations. CMZ did not slow the rate of recovery from in activation. In conclusion, CMZ inhibits Ca2+ channel current in a use-depen dent manner. This inhibition is independent of CaMK-II and other Ca2+/CaM-d ependent pathways. Therefore it is likely due to direct blockade of Ca2+ ch annels by CMZ. CMZ may reduce the outer surface charge and block the open s tate of the Ca2+ channels.