The spontaneous activity of pacemaker cells in the sino-atrial node (SAN) controls the heart rhythm and rate under physiological conditions. Pacemaker activity in SAN cells is due to the presence of the diastolic depolarization, a slow depolarization phase that drives the membrane voltage from the end of an action potential to the threshold of a new action potential. SAN cells express a wide array of ionic channels, but we have limited knowledge about their functional role in pacemaker activity and we still do not know which channels play a prominent role in the generation of the diastolic depolarization. It is thus important to provide genetic evidence linking the activity of genes coding for ionic channels to specific alterations of pacemaker activity of SAN cells. Here, we show that target inactivation of the gene coding for ␣1D (Cav1.3) Ca 2؉ channels in the mouse not only significantly slows pacemaker activity but also promotes spontaneous arrhythmia in SAN pacemaker cells. These alterations of pacemaker activity are linked to abolition of the major component of the L-type current (I Ca,L) activating at negative voltages. Pharmacological analysis of I Ca,L demonstrates that Cav1.3 gene inactivation specifically abolishes I Ca,L in the voltage range corresponding to the diastolic depolarization. Taken together, our data demonstrate that Ca v1.3 channels play a major role in the generation of cardiac pacemaker activity by contributing to diastolic depolarization in SAN pacemaker cells.

The modulation of voltage-dependent Ca2+ channels at presynaptic nerve terminals is an important factor in the control of neurotransmitter release and synaptic efficacy. Some terminals contain multiple Ca2(+)-channel subtypes (N and P/Q), which are differentially regulated by G-protein activation and by protein kinase C (PKC)-dependent phosphorylation. Regulation of channel activity by crosstalk between second messenger pathways has been reported although the molecular mechanisms underlying crosstalk have not been described. Here we show that crosstalk occurs at the level of the presynaptic Ca2(+)-channel complex. The alpha1 subunit domain I-II linker, which connects the first and second transmembrane domains, contributes to the PKC-dependent upregulation of channel activity, while G-protein-dependent inhibition occurs through binding of Gbetagamma to two sites in the I-II linker. Crosstalk results from the PKC-dependent phosphorylation of one of the Gbetagamma binding sites which antagonizes Gbetagamma-induced inhibition. The results provide a mechanism for the highly regulated and dynamic control of neurotransmitter release that depends on the integration of multiple presynaptic signals.

P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Here we report that alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants.

Analgesic therapies are still limited and sometimes poorly effective, therefore finding new targets for the development of innovative drugs is urgently needed. In order to validate the potential utility of blocking T-type calcium channels to reduce nociception, we explored the effects of intrathecally administered oligodeoxynucleotide antisenses, specific to the recently identified T-type calcium channel family (Ca V 3.1, Ca V 3.2, and Ca V 3.3), on reactions to noxious stimuli in healthy and mononeuropathic rats. Our results demonstrate that the antisense targeting Ca V 3.2 induced a knockdown of the Ca V 3.2 mRNA and protein expression as well as a large reduction of 'Ca V 3.2-like' T-type currents in nociceptive dorsal root ganglion neurons. Concomitantly, the antisense treatment resulted in major antinociceptive, anti-hyperalgesic, and anti-allodynic effects, suggesting that Ca V 3.2 plays a major pronociceptive role in acute and chronic pain states. Taken together, the results provide direct evidence linking Ca V 3.2 T-type channels to pain perception and suggest that Ca V 3.2 may offer a specific molecular target for the treatment of pain.