Transient and Big Are Key Features of an Invertebrate T-type Channel (LCa3) from the Central Nervous System of Lymnaea stagnalis

Here we describe features of the first non-mammalian T-type calcium channel (LCav3) expressed in vitro. This molluscan channel possesses combined biophysical properties that are reminiscent of all mammalian T-type channels. It exhibits T-type features such as “transient” kinetics, but the “tiny” label, usually associated with Ba2+ conductance, is hard to reconcile with the “bigness” of this channel in many respects. LCav3 is 25% larger than any voltage-gated ion channel expressed to date. It codes for a massive, 322-kDa protein that conducts large macroscopic currents in vitro. LCav3 is also the most abundant Ca2+ channel transcript in the snail nervous system. A window current at typical resting potentials appears to be at least as large as that reported for mammalian channels. This distant gene provides a unique perspective to analyze the structural, functional, drug binding, and evolutionary aspects of T-type channels. Here we describe features of the first non-mammalian T-type calcium channel (LCav3) expressed in vitro. This molluscan channel possesses combined biophysical properties that are reminiscent of all mammalian T-type channels. It exhibits T-type features such as “transient” kinetics, but the “tiny” label, usually associated with Ba2+ conductance, is hard to reconcile with the “bigness” of this channel in many respects. LCav3 is 25% larger than any voltage-gated ion channel expressed to date. It codes for a massive, 322-kDa protein that conducts large macroscopic currents in vitro. LCav3 is also the most abundant Ca2+ channel transcript in the snail nervous system. A window current at typical resting potentials appears to be at least as large as that reported for mammalian channels. This distant gene provides a unique perspective to analyze the structural, functional, drug binding, and evolutionary aspects of T-type channels. IntroductionT-type calcium channels open in response to slight depolarizations in the low voltage range. Paradoxically, they are also recruited after membrane hyperpolarization as occurs during rebound burst firing (1.Kim D. Song I. Keum S. Lee T. Jeong M.J. Kim S.S. McEnery M.W. Shin H.S. Neuron. 2001; 31: 35-45Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). A window current of T-type channels is a feature that permits Ca2+ entry at rest (2.Chemin J. Monteil A. Briquaire C. Richard S. Perez-Reyes E. Nargeot J. Lory P. FEBS Lett. 2000; 478: 166-172Crossref PubMed Scopus (95) Google Scholar) and contributes to differentiation and growth promoting functions in both excitable and non-excitable cells (3.Lory P. Bidaud I. Chemin J. Cell Calcium. 2006; 40: 135-146Crossref PubMed Scopus (111) Google Scholar). T-type channels are also a leading pharmaceutical drug target and are implicated in a wide range of conditions such as epilepsy, pain, hypertension, cancer, and mental disorders (4.Shin H.S. Cheong E.J. Choi S. Lee J. Na H.S. Curr. Opin. Pharmacol. 2008; 8: 33-41Crossref PubMed Scopus (76) Google Scholar).T-type Ca2+ currents were first measured in starfish eggs using a two-electrode voltage clamp (5.Hagiwara S. Ozawa S. Sand O. J. Gen. Physiol. 1975; 65: 617-644Crossref PubMed Scopus (162) Google Scholar). Currents conducted by “Channel I” were evoked by small depolarizations (low voltage-activated), visible as a small hump in a current amplitude versus test potential plot, appearing inconsequential beside the Channel II currents elicited by larger depolarizations (high voltage-activated). Ca2+ channel types would be discriminated further by Tsien and co-workers (6.Nowycky M.C. Fox A.P. Tsien R.W. Nature. 1985; 316: 440-443Crossref PubMed Scopus (1600) Google Scholar) on the basis of properties where Ba2+ is the charge carrier. High voltage-activated L-type channels have a large unitary Ba2+ conductance with long-lasting openings, N-type (or non-L-type) channels are typically associated with neurons of intermediate unitary conductance, and the low voltage-activated, T-type channels produce transient currents that are of tiny unitary conductance in Ba2+ and close slowly upon membrane repolarization, producing a slowly deactivating tail current (6.Nowycky M.C. Fox A.P. Tsien R.W. Nature. 1985; 316: 440-443Crossref PubMed Scopus (1600) Google Scholar).T-type channels remain as the least understood among the Ca2+ channel families. Although most of the 10 mammalian Ca2+ channel genes were characterized in the late 1980s, an additional decade was required for a description of the three T-type genes, Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I) (7.Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1320) Google Scholar). Progress in understanding T-type channel functions continues to be hampered by the lack of highly selective blockers that discriminate between Cav3 channel types or separate Cav3 channels from related L-type (Cav1) and non-L-type (Cav2) Ca2+ channels, which usually produce more robust Ca2+ entry into the same cells (7.Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1320) Google Scholar).Here we describe the in vitro expression characteristics of the first non-mammalian, T-type channel, LCav3, cloned from the pond snail, Lymnaea stagnalis. This structurally distant channel has quintessential features of T-types such as transient kinetics. LCav3 is big in many respects, such as its protein size; it expresses large macroscopic currents in human cells, it is the most abundant Ca2+ channel transcript in the snail nervous system, and it generates window currents that appear to be at least as large as those reported for mammalian channels. LCav3 provides a unique perspective to analyze the structure, function, and drug binding of T-type channels and serves as a useful surrogate in residue swapping experiments. Searches for the fundamental mechanisms that regulate this singleton invertebrate T-type channel will be facilitated by the simple molluscan preparation, where accessible and identified neurons underlying well described behaviors can be studied in isolated Lymnaea neurons, cultured synapses, or within intact, identified networks in situ. Also, LCav3 provides nourishment for evolutionary speculation. Although the first gastropods (500 million years ago) are likely quite distant from this ancestral branch point, the extant snail homolog, LCav3, is reminiscent of the gene that predates the speciation that led to the emergence of the three distinct, mammalian T-type channel genes. IntroductionT-type calcium channels open in response to slight depolarizations in the low voltage range. Paradoxically, they are also recruited after membrane hyperpolarization as occurs during rebound burst firing (1.Kim D. Song I. Keum S. Lee T. Jeong M.J. Kim S.S. McEnery M.W. Shin H.S. Neuron. 2001; 31: 35-45Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). A window current of T-type channels is a feature that permits Ca2+ entry at rest (2.Chemin J. Monteil A. Briquaire C. Richard S. Perez-Reyes E. Nargeot J. Lory P. FEBS Lett. 2000; 478: 166-172Crossref PubMed Scopus (95) Google Scholar) and contributes to differentiation and growth promoting functions in both excitable and non-excitable cells (3.Lory P. Bidaud I. Chemin J. Cell Calcium. 2006; 40: 135-146Crossref PubMed Scopus (111) Google Scholar). T-type channels are also a leading pharmaceutical drug target and are implicated in a wide range of conditions such as epilepsy, pain, hypertension, cancer, and mental disorders (4.Shin H.S. Cheong E.J. Choi S. Lee J. Na H.S. Curr. Opin. Pharmacol. 2008; 8: 33-41Crossref PubMed Scopus (76) Google Scholar).T-type Ca2+ currents were first measured in starfish eggs using a two-electrode voltage clamp (5.Hagiwara S. Ozawa S. Sand O. J. Gen. Physiol. 1975; 65: 617-644Crossref PubMed Scopus (162) Google Scholar). Currents conducted by “Channel I” were evoked by small depolarizations (low voltage-activated), visible as a small hump in a current amplitude versus test potential plot, appearing inconsequential beside the Channel II currents elicited by larger depolarizations (high voltage-activated). Ca2+ channel types would be discriminated further by Tsien and co-workers (6.Nowycky M.C. Fox A.P. Tsien R.W. Nature. 1985; 316: 440-443Crossref PubMed Scopus (1600) Google Scholar) on the basis of properties where Ba2+ is the charge carrier. High voltage-activated L-type channels have a large unitary Ba2+ conductance with long-lasting openings, N-type (or non-L-type) channels are typically associated with neurons of intermediate unitary conductance, and the low voltage-activated, T-type channels produce transient currents that are of tiny unitary conductance in Ba2+ and close slowly upon membrane repolarization, producing a slowly deactivating tail current (6.Nowycky M.C. Fox A.P. Tsien R.W. Nature. 1985; 316: 440-443Crossref PubMed Scopus (1600) Google Scholar).T-type channels remain as the least understood among the Ca2+ channel families. Although most of the 10 mammalian Ca2+ channel genes were characterized in the late 1980s, an additional decade was required for a description of the three T-type genes, Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I) (7.Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1320) Google Scholar). Progress in understanding T-type channel functions continues to be hampered by the lack of highly selective blockers that discriminate between Cav3 channel types or separate Cav3 channels from related L-type (Cav1) and non-L-type (Cav2) Ca2+ channels, which usually produce more robust Ca2+ entry into the same cells (7.Perez-Reyes E. Physiol. Rev. 2003; 83: 117-161Crossref PubMed Scopus (1320) Google Scholar).Here we describe the in vitro expression characteristics of the first non-mammalian, T-type channel, LCav3, cloned from the pond snail, Lymnaea stagnalis. This structurally distant channel has quintessential features of T-types such as transient kinetics. LCav3 is big in many respects, such as its protein size; it expresses large macroscopic currents in human cells, it is the most abundant Ca2+ channel transcript in the snail nervous system, and it generates window currents that appear to be at least as large as those reported for mammalian channels. LCav3 provides a unique perspective to analyze the structure, function, and drug binding of T-type channels and serves as a useful surrogate in residue swapping experiments. Searches for the fundamental mechanisms that regulate this singleton invertebrate T-type channel will be facilitated by the simple molluscan preparation, where accessible and identified neurons underlying well described behaviors can be studied in isolated Lymnaea neurons, cultured synapses, or within intact, identified networks in situ. Also, LCav3 provides nourishment for evolutionary speculation. Although the first gastropods (500 million years ago) are likely quite distant from this ancestral branch point, the extant snail homolog, LCav3, is reminiscent of the gene that predates the speciation that led to the emergence of the three distinct, mammalian T-type channel genes.

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.