The Ca 2؉ channel ␣1A-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca 2؉ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. ␣1A-Subunits are thought to support both P-and Q-type Ca 2؉ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca 2؉ delivery system at excitatory nerve terminals. We generated ␣1A-deficient mice (␣1A ؊͞؊ ) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying Ϸ3-4 weeks after birth. P-type currents in Purkinje neurons and P-and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca 2؉ channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in ␣1A ؊͞؊ hippocampal slices persisted despite the lack of P͞Q-type channels but showed enhanced reliance on N-type and R-type Ca 2؉ entry. The ␣1A ؊͞؊ mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in ␣1A. The ␣ 1A -subunit, the most abundant ␣ 1 -subunit in vertebrate brain (1), mediates Ca 2ϩ influx across presynaptic and somatodendritic membranes, thereby triggering fast neurotransmitter release and other key neuronal responses (2-5). Because of its high expression levels in the brain, the ␣ 1A -subunit was the first representative of its subclass to be isolated by cDNA cloning (1, 6). This predominantly neuronal subclass also includes ␣ 1B (N-type Ca 2ϩ channel) and ␣ 1E [possibly R-type Ca 2ϩ channel (7-9)] and is referred to as ABE or Ca V 2. There is no information to date on the behavioral or electrophysiological consequences of deleting a member of the ABE subfamily.␣ 1A Transcripts are widely distributed in rat (10) and human brain (11), most prominently in cell body layers in cerebellum and hippocampus. At the subcellular level, ␣ 1A immunoreactivity has been found in cell bodies, dendrites, and presynaptic terminals (12). Less clear has been the role of ␣ 1A in supporting Ca 2ϩ channel components defined by biophysical and pharmacological criteria. In either Xenopus oocytes (13, 14) or HEK293 cells (15), expression of ␣ 1A -subunits along with ancillary ␣ 2 ͞␦-and ␤-subunits generated currents with properties closely resembling the Q-type current found in cerebellar granule cells (8) and much less the P-type current first described in cerebellar Purkinje neurons by Llinás and colleagues (16,17). Unlike native P-type channels (18), the expressed currents showed pronounced inactivation during sustained depolarizations and responded to -agatoxin IVA (-Aga-IVA) at half-blocking doses of Ϸ100 nM, not Ϸ1 nM (13). Various explanations for the discrepancies have been advanced...

Functional presynaptic vesicles have been subdivided into readily releasable (RRP) and reserve (RP) pools. We studied recycling properties of RRP vesicles through differential retention of FM1-43 and FM2-10 and by varying the time window for FM dye uptake. Both approaches indicated that vesicles residing in the RRP underwent rapid endocytosis (tau approximately 1s), whereas newly recruited RP vesicles were recycled slowly (tau approximately 30 s). With repeated challenges (hypertonic or electrical stimuli), the ability to release neurotransmitter recovered 10-fold more rapidly than restoration of FM2-10 destaining. Finding neurotransmission in the absence of destaining implied that rapidly endocytosed RRP vesicles were capable of reuse, a process distinct from repopulation from the RP. Reuse would greatly expand the functional capabilities of a limited number of vesicles in CNS terminals, particularly during intermittent bursts of activity.

Several human channelopathies result from mutations in alpha1A, the pore-forming subunit of P/Q-type Ca2+ channels, conduits of presynaptic Ca2+ entry for evoked neurotransmission. We found that wild-type human alpha1A subunits supported transmission between cultured mouse hippocampal neurons equally well as endogenous mouse alpha1A, whereas introduction of impermeant human alpha1A hampered the effect of endogenous subunits. Thus, presynaptic P/Q-type channels may compete for channel type-preferring "slots" that limit their synaptic effectiveness. The existence of slots generates predictions for how neurotransmission might be affected by changes in Ca2+ channel properties, which we tested by studying alpha1A mutations that are associated with familial hemiplegic migraine type 1 (FHM1). Mutant human P/Q-type channels were impaired in contributing to neurotransmission in precise accord with their deficiency in supporting whole-cell Ca2+ channel activity. Expression of mutant channels in wild-type neurons reduced the synaptic contribution of P/Q-type channels, suggesting that competition for type-preferring slots might support the dominant inheritance of FHM1.

Transmission at the mouse neuromuscular junction normally relies on P͞Q-type channels, but became jointly dependent on both Nand R-type Ca 2؉ channels when the P͞Q-type channel ␣1A subunit was deleted. R-type channels lay close to Ca 2؉ sensors for exocytosis and I K(Ca) channel activation, like the P͞Q-type channels they replaced. In contrast, N-type channels were less well localized, but abundant enough to influence secretion strongly, particularly when action potentials were prolonged. Our data suggested that active zone structures may select among multiple Ca 2؉ channels in the hierarchy P͞Q>R>N. The ␣1A؊͞؊ neuromuscular junction displayed several other differences from wild-type: lowered quantal content but greater ability to withstand reductions in the Ca 2؉ ͞Mg 2؉ ratio, and little or no paired-pulse facilitation, the latter findings possibly reflecting compensatory mechanisms at individual release sites. Changes in presynaptic function were also associated with a significant reduction in the size of postsynaptic acetylcholine receptor clusters. neuromuscular junction ͉ ␣1A knockout ͉ paired-pulse facilitation ͉ SNX-482 ͉ Ca 2ϩ -activated potassium channel