Are Cav1.3 pacemaker channels in chromaffin cells? Possible bias from resting cell conditions and DHP blockers usage

Mouse and rat chromaffin cells (MCCs, RCCs) fire spontaneously at rest and their activity is mainly supported by the two L-type Ca2+ channels expressed in these cells (Ca(v)1.2 and Ca(v)1.3). Using Ca(v)1.3(-/-) KO MCCs we have shown that Ca-v 1.3 possess all the prerequisites for carrying subthreshold currents that sustain low frequency cell firing near resting (0.5 to 2 Hz at-50 mV):(1) low-threshold and steep voltage dependence of activation, slow and incomplete inactivation during pulses of several hundreds of milliseconds. Ca(v)1.2 contributes also to pacemaking MCCs and possibly even Na+ channels may participate in the firing of a small percentage of cells. We now show that at potentials near resting (-50 mV), Ca(v)1.3 carries equal amounts of Ca2+ current to Ca(v)1.2 but activates at 9 mV more negative potentials. MCCs express only TTX-sensitive Na(v)1 channels that activate at 24 mV more positive potentials than Ca(v)1.3 and are fully inactivating. Their blockade prevents the firing only in a small percentage of cells (13%). This suggests that the order of importance with regard to pacemaking MCCs is: Ca(v)1.3, Ca(v)1.2 and Na(v)1. The above conclusions, however, rely on the proper use of DHPs, whose blocking potency is strongly holding potential dependent. We also show that small increases of KCl concentration steadily depolarize the MCCs causing abnormally increased firing frequencies, lowered and broadened AP waveforms and an increased facility of switching "non-firing" into "firing" cells that may lead to erroneous conclusions about the role of Ca(v)1.3 and Ca(v)1.2 as pacemaker channels in MCCs.(2)