Calcium channels are 'Unblocked' within a few milliseconds after photoremoval of nifedipine

The organic Ca²⁺ antagonists are potent inhibitors of Ca²⁺ influx in cardiac and smooth muscle and are widely used clinically in the treatment of various cardiovascular disorders [4, 11]. It appears that Ca²⁺ antagonist binding prevents the normal movement of ions through Ca²⁺ channels, perhaps via an open-channel blockade [1] mechanism. Although this concept is generally accepted, questions do remain about the detailed relationship between binding and blockade in the case of the structurally diverse organic Ca²⁺ channel blockers [4, 6-8, 10, 11, 13, 15]; e.g., (1) do they bind preferentially to open, closed and/or inactivated channels; (2) are there multiple binding sites; (3) do they act at extracellular and/or intracellular sites; and (4) does blocking or unblocking depend on membrane potential or its history? The dihydropyridine Ca²⁺ antagonist, nifedipine, contains an o-nitrobenzyl moiety and is photolabile [3]; irradiation yields a molecule devoid of channel blocking activity [3, 9, 15] and the photoconversion reactions are complete within 100 μs [9]. Taking advantage of these properties to study the mechanistic details of nifedipine blockade of Ca²⁺ channels, we examined the waveform of the slow inward Ca²⁺ current (I_si) in atrial fibers before and following flash-induced removal of nifedipine. After flashes, we find that nifedipine blockade is reversed within at most a few milli-seconds and that the rate of I_si reactivation parallels the normal, voltage-dependent activation rate. Our results imply that nifedipine binds to and stabilizes resting, closed Ca²⁺ channels and are not in agreement with the recent conclusions of Morad and coworkers [9] that photoconversion of nifedipine must be followed by membrane repolarization in order to effect recovery of I_si and tension. In addition, we find that the rate of I_si decay is increased transiently following flashes and we suggest that this effect may account for the apparent voltage dependence of recovery reported previously [9].