Cytoplasmic Bkca Channel Intron-Containing Mrnas Contribute to the Intrinsic Excitability of Hippocampal Neurons
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Cytoplasmic BKCa channel intron-containing mRNAs contribute to the intrinsic excitability of hippocampal neurons Thomas J. Bell*, Kevin Y. Miyashiro*, Jai-Yoon Sul*†, Ronald McCullough‡, Peter T. Buckley*§, Jeanine Jochems*, David F. Meaney¶, Phil Haydon†, Charles Cantorʈ**, Thomas D. Parsons††‡‡, and James Eberwine*§** §Penn Genome Frontiers Institute, and Departments of *Pharmacology, †Neuroscience, ¶Bioengineering, and ††Otorhinolaryngology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; ‡Program of Molecular and Cellular Biology and Biochemistry, Boston University, Boston, MA 02215; ʈSequenom, Inc., 3595 John Hopkins Court, San Diego, CA 92121; and ‡‡Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348 Contributed by Charles Cantor, December 17, 2007 (sent for review December 5, 2007) -High single-channel conductance K؉ channels, which respond is not fully resolved, but the expression and subcellular distri jointly to membrane depolarization and micromolar concentra- bution patterns of splice variants are expected to be one func- tions of intracellular Ca2؉ ions, arise from extensive cell-specific tionally significant factor in determining their characteristic alternative splicing of pore-forming ␣-subunit mRNAs. Here, we input–output properties. Indeed, there is some evidence for ϩ report the discovery of an endogenous BKCa channel ␣-subunit members of the K channel family, for example BKCa (12) or intron-containing mRNA in the cytoplasm of hippocampal neurons. A-type (13), establishing functional gradients within the den- This partially processed mRNA, which comprises Ϸ10% of the total dritic processes of mature neurons, where they transform the BKCa channel ␣-subunit mRNAs, is distributed in a gradient shape of local synaptic potentials or the size of somatic action throughout the somatodendritic space. We selectively reduced potentials (14). endogenous cytoplasmic levels of this intron-containing transcript Here we report a novel form of posttranscriptional gene by RNA interference without altering levels of the mature splice expression (cytoplasmic splicing of a RNA with a retained forms of the BKCa channel mRNAs. In doing so, we could demon- intron) that plays a role in generating local populations of BKCa strate that changes in a unique BKCa channel ␣-subunit intron- channel mRNAs and proteins in hippocampal neurons. These containing splice variant mRNA can greatly impact the distribution results show that the regulation of a cytoplasmic BKCa channel of the BKCa channel protein to dendritic spines and intrinsic firing intron-containing mRNAs partially underlie the characteristic properties of hippocampal neurons. These data suggest a new input–output properties of hippocampal neurons. regulatory mechanism for modulating the membrane properties and ion channel gradients of hippocampal neurons. Results BKCa Channel Intron-Containing mRNAs Are Present in Hippocampal dendrite ͉ epilepsy ͉ intron-retention ͉ KCNMA1 ͉ local splicing Dendrites. To determine the repertoire of mRNA splice variants in the postsynaptic compartment, we prepared a cDNA template derived from antisense RNA amplification of polyA mRNA n mammalian neurons, the firing of an action potential re- isolated from rat hippocampal dendrites, which were harvested quires the coordinated gating of at least a dozen different I from 14- to 21-day-old cultures in vitro (15). Dendrites were classes of voltage-gated ion channels. The integration of these carefully harvested individually to ensure that no cell bodies, and currents manifests itself as characteristic input–output proper- thus nuclei, of any neuron or nonneuronal cell were harvested. ties intrinsic to each neuron. One such firing property of some From this starting material, we detected two separate PCR neurons, such as those in the hippocampus (1, 2) or cerebellum products using primers specific to intron 16 of KCNMA1 [sup- (3), is the ability to initiate repetitive burst firing in response to porting information (SI) Fig. 5a]. Intron 16 (i16) is Ϸ6,000 nt depolarizing current. Voltage-clamp analysis suggests that the long and lies upstream of a previously described ‘‘hot spot’’ for small net inward current that drives the depolarizing momentum alternative exon usage (16). Exons surrounding i16 encode the is a result of a subtle balance of the sum of inward and outward 3Ј-end of the first of two highly conserved Ca2ϩ-binding RCK postspike currents (4). Although short-term and long-term (regulators of conductance Kϩ channel) domains and the non- feedback mechanisms exist to preserve burst firing (5), it is conserved linker region spanning the first and second RCK known that relatively small changes in the size of an individual module (17) (see Fig. 1a). The nucleotide sequence of the PCR current may have a dramatic impact on firing activity (6). products (330 and 390 nt) proved to be identical to the rat One transient current activated during the falling phase of the genomic BKCa channel genomic DNA sequence deposited in action potential is the BKCa channel. In the central nervous GenBank. We also observed the presence and absence of introns system, BKCa channels are localized to the cell soma as well as in other mRNAs in amplified polyA mRNA dendrite samples the pre- and postsynaptic terminals of neurons, where they regulate fundamental neuronal functions such as burst firing, neurotransmitter release, shaping action potential waveforms, Author contributions: T.J.B. and K.Y.M. contributed equally to this work; T.J.B., K.Y.M., and frequency tuning (7). Native channels are assembled as J.-Y.S., R.M., P.T.B., C.C., T.D.P., and J.E. designed research; T.J.B., K.Y.M., J.-Y.S., R.M., P.T.B., tetramers of the pore-forming ␣-subunits encoded by a single and J.J. performed research; T.J.B., K.Y.M., J.-Y.S., R.M., P.T.B., D.F.M., P.H., C.C., and J.E. contributed new reagents/analytic tools; T.J.B., K.Y.M., J.-Y.S., R.M., P.T.B., C.C., T.D.P., and gene, KCNMA1 (8), which is subject to vast tissue-specific (9) J.E. analyzed data; and T.J.B., K.Y.M., J.-Y.S., R.M., P.T.B., J.J., D.F.M., P.H., C.C., T.D.P., and and cell-specific alternative splicing (10). The resulting func- J.E. wrote the paper. tional heterogeneity in BKCa channel currents is due in part to The authors declare no conflict of interest. 2ϩ these splicing events generating BKCa channels with altered Ca **To whom correspondence may be addressed. E-mail: [email protected] or sensitivity and gating kinetics (7) as well as altered channel [email protected]. trafficking (11). Channel differences also arise from modulation This article contains supporting information online at www.pnas.org/cgi/content/full/ via a family of tissue-specific auxiliary -subunits (10). Cur- 0711796105/DC1. CELL BIOLOGY rently, the functional diversity among BKCa channels in neurons © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711796105 PNAS ͉ February 12, 2008 ͉ vol. 105 ͉ no. 6 ͉ 1901–1906 Downloaded by guest on October 1, 2021 products. The i16-containing transcript represented 10.3% Ϯ 2.3% of the total BKCa channel transcript population (Fig. 1a). The levels of i16-containing transcripts reported here are in agreement with a previous study showing other KCNMA1 splice variants typically represent a small subset (Ϸ10%) of the total KCNMA1 transcript population (19). We next determined the endogenous KCNMA1 exonic com- bination between i16 to e18 in both hippocampal tissue and isolated dendrites. This region consists of four downstream exons: two constitutive exons (e17 and e18) and two alternatively spliced exons (e17a and e17b). Here we amplified cDNA with two sets of PCR primers: (i) e16 forward and e18 reverse to capture ‘‘all BKCa channel transcripts’’ or (ii) i16–1 forward and e18 reverse to capture ‘‘only i16-containg transcripts’’ (Fig. 1a). The PCR products were simultaneously assayed with the two different extension primers (XP2 and XP3) to capture all four exonic combinations (Fig. 1a). In the ‘‘all BKCa channel tran- scripts’’ population, every exonic combination was detected, but two highly abundant variants, e17–e18 and e17–e17a–e18, made up the bulk of the BKCa channel transcript population (Fig. 1a). A remarkably different pattern of splicing was detected in the ‘‘only i16-containing transcript’’ population. Unexpectedly, only one splice variant was detected; all of the i16-containing tran- scripts skip e17a and e17b and splice e17 directly to e18 (Fig. 1a). These data show that i16-containing mRNAs are restricted to a subset of the KCNMA1 transcript population containing cor- rectly spliced downstream exons, e17 and e18. Furthermore, Fig. 1. BKCa channel intron-containing mRNAs are present in the cytoplasm these results highlight two key properties of i16-contiaining of hippocampal neurons. (a) Putative membrane topology of the BKCa channel transcripts. First, they are present at biologically significant ␣-subunit and the location of two sites of alternative splicing (black circles) are levels in the hippocampus, and, more surprisingly, the overall shown. Exons are denoted with vertical solid bars and introns with horizontal complexity of the i16-contiaining transcripts transcript popula- lines. Exon lengths are 69, 12, 87, 77, 9, 174, and 94 nt for e15, e15a, e16, e17, tion is less diverse then would be possible if all combinations of e17a, e17b, and e18, respectively. Intron lengths are 2,340, 6,222, 6,654, 4,528, 6,421, and 30,920 nt for i15, i15a, i16-containing, i17, i17a, and i17b, respec- the downstream exons were associated with the retained intron. tively. KCNMA1 splice variant analysis was performed in hippocampal tissue. These data show the abundance of four KCNMA1 splice variants and the The Subcellular Localization of BKCa Channel and the i16-Containing exonic linkage analysis of each splice variant to i16-containing region.