In Vitro Characterization of L-Type Calcium Channels and Their Contribution to Firing Behavior in Invertebrate Respiratory Neurons

Abstract

L-type calcium channel activity has been associated with a number of cytoplasmic responses, including gene transcription and activation of calcium-dependent enzymes, yet their direct contribution to the electrical activities of neurons has remained largely unexplored. Here we report the cloning and functional characterization of a molluscan L-type calcium channel homologue, LCa(v)1, and investigate its role in coordinating neuronal firing patterns. The LCav1 channel exhibits many hallmarks of vertebrate L-type channels in that it is high-voltage activated, slowly inactivating, and dihydropyridine sensitive and displays calcium-dependent inactivation in recording solutions with standard EGTA concentrations. We show that despite comprising less than approximately 20% of the total whole cell current in identified Lymnaea respiratory network neurons, the L-type channels are essential for maintaining rhythmic action potential discharges without being involved in synaptic release. Our data therefore suggest an important role of L-type calcium channels in maintaining rhythmical pattern activity underlying breathing behavior in Lymnaea.

PKC-Induced Intracellular Trafficking of CaV2 Precedes Its Rapid Recruitment to the Plasma Membrane

“…As a further test for specificity of this antibody, we transfected mammalian GripTite 293 MSR cells with the Lymnaea Ca V 2 channel (Spafford et al, 2003;Spafford et al, 2004), which differs from the Aplysia Ca V 2 channel in only 2 of the 18 aa of the sequence against which the antibody was generated [SEHYNQPEWHVQFLYITE (Lymnaea) vs SEHYNQPEWFVDFLYITE (Aplysia)]. Cells were also transfected with the full-length Lymnaea Ca V 1 channel (Spafford et al, 2006). These channels were expressed in the pIRES2 bicistronic vector, where EGFP is coupled to the mRNA of LCa V 2 and LCa V 1, allowing ready visualization of transfected cells.…”

“…Cells were transfected using the calcium phosphate method with 6.7 g of either Lymnaea stagnalis LCa V 1 channel (Spafford et al, 2006) or LCa V 2 channel (Spafford et al, 2003) ␣ 1 subunits in phosphorylated internal ribosomal entry site-enhanced green fluorescent protein (pIRES2-EGFP) vector (Clontech Laboratories, Mountain View, CA) plus 6.7 g of rat ␤ 1 subunit in pMT2 and 6.7 g of rat ␣ 2 ␦ in pMT2. Transfected cells were subsequently washed with PBS and detached using trypsin-EDTA solution (Sigma).…”

G-proteins modulate invertebrate synaptic calcium channel (LCav2) differently from the classical voltage-dependent regulation of mammalian Cav2.1 and Cav2.2 channels

“…To study the characteristics of the LCa v 2 calcium channel, the wildtype LCa v 2 gene was cloned into pIRES2-EGFP vector from a previously modified LCa v 2 construct, dubbed 5ЈRatCa v 2.1-LCa v 2, in pMT2 plasmid, which bears an N-terminus from mammalian Ca v 2.1 (Spafford et al, 2003a;Spafford et al, 2004;Spafford et al, 2006). The wild-type channel was reconstructed with the Nterminus of wild-type LCa v 2 (GenBank Accession No.…”

“…Shown in Fig.1A is a gene tree illustrating the relationships of human homologs to an identified set of calcium channel subunit genes from Lymnaea stagnalis, all previously expressed in human cell lines: LCa v 1 (Spafford et al, 2006), LCa v 2 (Spafford et al, 2003a) and LCa v 3 (Senatore and Spafford, 2010). Ca v 3 channels such as LCa v 3 bear transient, T-Type currents that are low voltageactivated (LVA) and open at resting membrane potentials (Senatore and Spafford, 2010).…”

Trophic Factor-Induced Intracellular Calcium Oscillations Are Required for the Expression of Postsynaptic Acetylcholine Receptors during Synapse Formation betweenLymnaeaNeurons

“…2؉ oscillations required Ca 2؉ influx through VGCCs To determine whether CM-induced Ca 2ϩ oscillations involved Ca 2ϩ influx through VGCCs, the LPeD1 neurons were exposed to CM either in the presence of Ca 2ϩ channel blocker cadmium (Cd 2ϩ , 100 M) or nifedipine (10 M) shown previously to essentially block L-type Ca 2ϩ channels in Lymnaea neurons (Spafford et al, 2006). We found that CM-induced Ca 2ϩ increase in LPeD1 was gradually and completely blocked by Cd 2ϩ (n ϭ 8) (Fig.…”