The molecular diversity of voltage-activated calcium channels was established by studies showing that channels could be distinguished by their voltage-dependence, deactivation and single-channel conductance. Low-voltage-activated channels are called 'T' type because their currents are both transient (owing to fast inactivation) and tiny (owing to small conductance). T-type channels are thought to be involved in pacemaker activity, low-threshold calcium spikes, neuronal oscillations and resonance, and rebound burst firing. Here we report the identification of a neuronal T-type channel. Our cloning strategy began with an analysis of Genbank sequences defined as sharing homology with calcium channels. We sequenced an expressed sequence tag (EST), then used it to clone a full-length complementary DNA from rat brain. Northern blot analysis indicated that this gene is expressed predominantly in brain, in particular the amygdala, cerebellum and thalamus. We mapped the human gene to chromosome 17q22, and the mouse gene to chromosome 11. Functional expression of the channel was measured in Xenopus oocytes. Based on the channel's distinctive voltage dependence, slow deactivation kinetics, and 7.5-pS single-channel conductance, we conclude that this channel is a low-voltage-activated T-type calcium channel.

Voltage-activated Ca2+ channels exist as multigene families that share common structural features. Different Ca2+ channels are distinguished by their electrophysiology and pharmacology and can be classified as either low or high voltage-activated channels. Six alpha1 subunit genes cloned previously code for high voltage-activated Ca2+ channels; therefore, we have used a database search strategy to identify new Ca2+ channel genes, possibly including low voltage-activated (T-type) channels. A novel expressed sequence-tagged cDNA clone of alpha1G was used to screen a cDNA library, and in the present study, we report the cloning of alpha1H (or CavT.2), a low voltage-activated Ca2+ channel from human heart. Northern blots of human mRNA detected more alpha1H expression in peripheral tissues, such as kidney and heart, than in brain. We mapped the gene, CACNA1H, to human chromosome 16p13.3 and mouse chromosome 17. Expression of alpha1H in HEK-293 cells resulted in Ca2+ channel currents displaying voltage dependence, kinetics, and unitary conductance characteristic of native T-type Ca2+ channels. The alpha1H channel is sensitive to mibefradil, a nondihydropyridine Ca2+ channel blocker, with an IC50 of 1.4 micromol/L, consistent with the reported potency of mibefradil for T-type Ca2+ channels. Together with alpha1G, a rat brain T-type Ca2+ channel also cloned in our laboratory, these genes define a unique family of Ca2+ channels.

Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of nucleic acids. Since its introduction, real-time quantitative PCR has revolutionized the field of molecular diagnostics and the technique is being used in a rapidly expanding number of applications. This exciting technology has enabled the shift of molecular diagnostics toward a high-throughput, automated technology with lower turnaround times. This article reviews the basic principles of real-time PCR and describes the various chemistries available: the double-stranded DNA-intercalating agent SYBR Green 1, hydrolysis probes, dual hybridization probes, molecular beacons and scorpion probes. Quantitation methods are discussed in addition to the competing instruments available on the market. Examples of applications of this important and versatile technique are provided throughout the review.