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.

Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (␣ 1G , ␣ 1H , and ␣ 1I ). Northern blot and reverse transcriptase-polymerase chain reaction analyses show ␣ 1G , ␣ 1H , and ␣ 1I to be expressed throughout the newborn and juvenile rat brain. In contrast, while ␣ 1G and ␣ 1H mRNA are expressed in all regions in adult rat brain, ␣ 1I mRNA expression is restricted to the striatum. Expression of ␣ 1G , ␣ 1H , and ␣ 1I subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of ␣ 1G , ␣ 1H , and ␣ 1I under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of ␣ 1G , ␣ 1H , and ␣ 1I T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.

T-type calcium channels play critical roles in cellular excitability and have been implicated in the pathogenesis of a variety of neurological disorders including epilepsy. Although there have been reports that certain neuroleptics that primarily target D2 dopamine receptors and are used to treat psychoses may also interact with T-type Ca channels, there has been no systematic examination of this phenomenon. In the present paper we provide a detailed analysis of the effects of several widely used neuroleptic agents on a family of exogenously expressed neuronal T-type Ca channels (alpha1G, alpha1H, and alpha1I subtypes). Among the neuroleptics tested, the diphenylbutylpiperidines pimozide and penfluridol were the most potent T-type channel blockers with Kd values (approximately 30-50 nm and approximately 70-100 nm, respectively), in the range of their antagonism of the D2 dopamine receptor. In contrast, the butyrophenone haloperidol was approximately 12- to 20-fold less potent at blocking the various T-type Ca channels. The diphenyldiperazine flunarizine was also less potent compared with the diphenylbutylpiperadines and preferentially blocked alpha1G and alpha1I T-type channels compared with alpha1H. The various neuroleptics did not significantly affect T-type channel activation or kinetic properties, although they shifted steady-state inactivation profiles to more negative values, indicating that these agents preferentially bind to channel inactivated states. Overall, our findings indicate that T-type Ca channels are potently blocked by a subset of neuroleptic agents and suggest that the action of these drugs on T-type Ca channels may significantly contribute to their therapeutic efficacy.

1 This study examined the action of gabapentin (gabapentin,1-(aminomethyl) 2+ in¯ux were signi®cantly reduced by *25-30% in the presence of gabapentin (25 mM). 4 Gabapentin potently inhibited the peak whole-cell Ca 2+ channel current (I Ba ) in a dosedependent manner with an estimated IC 50 value of 167 nM. Block was incomplete and saturated at a maximal concentration of 25 mM. 5 Inhibition was signi®cantly decreased in the presence of the neutral amino acid L-isoleucine (25 mM) but unaected by application of the GABA B antagonist, saclofen (200 mM), suggesting a direct action on the a 2 d subunit of the Ca 2+ channel. 6 Gabapentin inhibition was voltage-dependent, producing an *7 mV hyperpolarizing shift in current voltage properties and reducing a non-inactivating component of whole-cell current activated at relatively depolarized potentials. 7 The use of speci®c Ca 2+ channel antagonists revealed a mixed pharmacology of the gabapentinsensitive current (N-, L-and P/Q-type), which is dominated by N-type current. 8 The present study is the ®rst to demonstrate that gabapentin directly mediates inhibition of voltage-gated Ca 2+ in¯ux in DRG neurones, providing a potential means for gabapentin to eectively mediate spinal anti-nociception.

Spatial and temporal changes in intracellular calcium concentrations are critical for controlling gene expression in neurons. In many neurons, activity-dependent calcium influx through L-type channels stimulates transcription that depends on the transcription factor CREB by activating a calmodulin-dependent pathway. Here we show that selective influx of calcium through P/Q-type channels is responsible for activating expression of syntaxin-1A, a presynaptic protein that mediates vesicle docking, fusion and neurotransmitter release. The initial P/Q-type calcium signal is amplified by release of calcium from intracellular stores and acts through phosphorylation that is dependent on the calmodulin-dependent kinase CaM K II/IV, protein kinase A and mitogen-activated protein kinase kinase. Initiation of syntaxin-1A expression is rapid and short-lived, with syntaxin-1A ultimately interacting with the P/Q-type calcium channel to decrease channel availability. Our results define an activity-dependent feedback pathway that may regulate synaptic efficacy and function in the nervous system.

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We have synthesized iodinated resiniferatoxin bearing a 4-hydroxy-5-iodo-3-methoxyphenylacetate ester (I-RTX) and have characterized its activity on rat and human TRPV1 (VR1) receptors, as well as in behavioral assays of nociception. In whole cell patch-clamp recordings from transfected cells the functional activity of I-RTX was determined. Currents activated by capsaicin exhibited characteristic outward rectification and were antagonized by capsazepine and I-RTX. On rat TRPV1 the affinity of I-RTX was 800-fold higher than that of capsazepine (IC 50 ϭ 0.7 and 562 nM, respectively) and 10-fold higher on rat versus human receptors (IC 50 ϭ 0.7 and 5.4 nM, respectively). The same difference was observed when comparing the inhibition of [ 3 H]RTX binding to rat and human TRPV1 membranes for both RTX and I-RTX. Additional pharmacological differences were revealed using protons as the stimulus. Under these conditions capsazepine only partly blocked currents through rat TRPV1 receptors (by 70 to 80% block), yet was a full antagonist on human receptors. In contrast, I-RTX completely blocked proton-induced currents in both species and that activated by noxious heat. I-RTX also blocked capsaicin-induced firing of C-fibers in a rat in vitro skin-nerve assay. Despite this activity and the high affinity of I-RTX for rat TRPV1, only capsazepine proved to be an effective antagonist of capsaicin-induced paw flinching in rats. Thus, although I-RTX has limited utility for in vivo behavioral studies it is a high-affinity TRPV1 receptor antagonist that will be useful to characterize the functional properties of cloned and native vanilloid receptor subtypes in vitro.The transient receptor potential TRPV1 vanilloid receptor (also known as VR1; see revised TRP channel nomenclature Montell et al., 2002) gates a nonselective cation channel that is expressed by sensory neurons and that can be activated by protons, heat, and capsaicin, the pungent ingredient of chili peppers (Caterina et al., 1997;Tominaga et al., 1998). Ligands acting at the TRPV1 vanilloid receptor subtype have the potential therapeutic utility to treat thermal hyperalgesia-related pain and some inflammatory conditions (for review, see Szallasi and Blumberg, 1999;Caterina and Julius, 2001). One of the first antagonists described for the capsaicin receptor was capsazepine (Bevan et al., 1992). This ligand has been used widely to explore the functional significance of TRPV1 receptors in pain. However it has relatively low micromolar affinity for TRPV1 receptors and because it also blocks voltage-gated calcium channels, this has made interpretation of functional data with this compound less straightforward (Docherty et al., 1997).To date, one of the highest affinity ligands reported for the TRPV1 receptor is the natural plant product resiniferatoxin (RTX), which was first isolated from Euphorbia resinifera (Szallasi and Blumberg, 1989). Interestingly, it has been shown recently that iodination of this agonist RTX confers antagonist-like properties to the ligand wit...

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