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.

Low voltage-activated Ca 2ϩ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy. Here we describe the cloning and expression of the third member of the T-type family, ␣1I or Ca v T.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopus oocytes or stably transfected human embryonic kidney-293 cells revealed novel gating properties. We compared these electrophysiological properties to those of the cloned T-type channels ␣1G and ␣1H and to the high voltage-activated channels formed by ␣1E␤ 3 . The ␣1I channels opened after small depolarizations of the membrane similar to ␣1G and ␣1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca 2ϩ injector. In oocytes, the kinetics were even slower, suggesting that components of the expression system modulate its gating properties. Steady-state inactivation occurred at higher potentials than any of the other T channels, endowing the channel with a substantial window current. The ␣1I channel could still be classified as T-type by virtue of its criss-crossing kinetics, its slow deactivation (tail current), and its small (11 pS) conductance in 110 mM Ba 2ϩ solutions. Based on its brain distribution and novel gating properties, we suggest that ␣1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

Tetrodotoxin (TTX)-sensitive Na currents were examined in single dissociated ventricular myocytes from neonatal rats. Single channel and whole cell currents were measured using the patch-clamp method . The channel density was calculated as 2/jrn1, which agreed with our usual finding of four channels per membrane patch . At 20°C, the single channel conductance was 20 pS . The open time distributions were fit by a single-exponential function with a mean open time of -., 1 .0 ms at membrane potentials from -60 to -40 mV. Averaged single channel and whole cell currents were similar when scaled and showed both fast and slow rates of inactivation . The inactivation and activation gating shifted quickly to hyperpolarized potentials for channels in cell-attached as well as excised patches, whereas a much slower shift occurred in whole cells. Slowly inactivating currents were present in both whole cell and single channel current measurements at potentials as positive as -40 mV. In whole cell measurements, the potential range could be extended, and slow inactivation was present at potentials as positive as -10 mV. The curves relating steady state activation and inactivation to membrane potential had very little overlap, and slow inactivation occurred at potentials that were positive to the overlap. Slow inactivation is in this way distinguishable from the overlap or window current, and the slowly inactivating current may contribute to the plateau of the rat cardiac action potential. On rare occasions, a second set of Na channels having a smaller unit conductance and briefer duration was observed . However, a separate set of threshold channels, as described by Gilly and Armstrong (1984 . Nature [Loud.] . 309:448), was not found. For the commonly observed Na channels, the number of openings in some samples far exceeded the number of channels per patch and the latencies to first opening or waiting times were not sufficiently dispersed to account for the slowly inactivating currents ; the slow inactivation was produced by channel reopening. A general model was developed to predict the number of openings in each sample . Models in which the number of openings per sample was due to a dispersion of waiting times combined with a rapid transition from an open to