Abstract
Voltage-gated Ca(2+) channels (Cav) undergo extensive alternative splicing that greatly enhances their functional diversity in excitable cells. Here, we characterized novel splice variants of the cytoplasmic C-terminal domain of Cav1.4 Ca(2+) channels that regulate neurotransmitter release in photoreceptors in the retina. These variants lack a portion of exon 45 and/or the entire exon 47 (Cav1.4Δex p45, Cav1.4Δex 47, Cav1.4Δex p45,47) and are expressed in the retina of primates but not mice. Although the electrophysiological properties of Cav1.4Δex p45 are similar to those of full-length channels (Cav1.4FL), skipping of exon 47 dramatically alters Cav1.4 function. Deletion of exon 47 removes part of a C-terminal automodulatory domain (CTM) previously shown to suppress Ca(2+)-dependent inactivation (CDI) and to cause a positive shift in the voltage dependence of channel activation. Exon 47 is crucial for these effects of the CTM because variants lacking this exon show intense CDI and activate at more hyperpolarized voltages than Cav1.4FL The robust CDI of Cav1.4Δex 47 is suppressed by CaBP4, a regulator of Cav1.4 channels in photoreceptors. Although CaBP4 enhances activation of Cav1.4FL, Cav1.4Δex 47 shows similar voltage-dependent activation in the presence and absence of CaBP4. We conclude that exon 47 encodes structural determinants that regulate CDI and voltage-dependent activation of Cav1.4, and is necessary for modulation of channel activation by CaBP4.
Highlights
In the retina, voltage-gated Cav1.4 (L-type) Ca2ϩ channels are localized in the synaptic terminals of rod and cone photoreceptors where they mediate Ca2ϩ signals that trigger glutamate release at the first synapse in the visual pathway [1]
To gain insights into how alternative splicing affects Cav1.4 channels containing 2ϫ13 and ␣2␦4, we analyzed the electrophysiological properties of new Cav1.4 splice variants that we discovered while isolating cDNAs encoding Cav1.4 from human retina
Unlike Cav1.4 ex43*, which lacks the C-terminal 256 amino acids of the channel [23], these variants lack only exon 47, which deletes part of the C-terminal automodulatory domain (CTM), but leaves the remaining C-terminal domain intact. We show that these variants are expressed at significant levels in human retina, and exhibit hyperpolarized voltages of activation and Ca2؉-dependent inactivation (CDI) similar to Cav1.4 ex43*
Summary
Identification of Novel Cav1.4 Distal C-terminal Splice Variants in Human Retina—Using RT-PCR to generate a cDNA construct corresponding to human Cav1.4 from retinal RNA, we amplified a shorter product than expected with primers flanking nucleotides 3913 to 5934 of the cDNA encoding the full-length channel (Cav1.4FL). Sequencing revealed that this fragment lacked the first 21 amino acids of exon 45 (p45) due to an alternative 3Ј splice site in exon 45. Cav1.4FL currents showed little inactivation during 1-s depolarizations regardless of whether Ca2ϩ or
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