Antisense oligonucleotides against α _1E reduce R-type calcium currents in cerebellar granule cells

Abstract

Many neurons of the central nervous system display multiple high voltage-activated Ca 2؉ currents, pharmacologically classified as L-, N-, P-, Q-, and R-type. Of these current types, the R-type is the least understood. The leading candidate for the molecular correlate of R-type currents in cerebellar granule cells is the ␣ 1E subunit, which yields Ca 2؉ currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against ␣ 1E could decrease the expression of R-type current in rat cerebellar granule neurons in culture. Cells were supplemented with either antisense or sense oligonucleotides and whole-cell patch clamp recordings were obtained after 6-8 days in vitro. Incubation with ␣ 1E antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from ؊10 ؎ 0.6 picoamperes͞picofarad (pA͞pF) (n ‫؍‬ 6) in the untreated controls to ؊4.8 ؎ 0.8 pA͞pF (n ‫؍‬ 11) (P < 0.01). In contrast, no significant changes in the current expression were seen in sense oligonucleotide-treated cells (؊11.3 ؎ 3.2 pA͞pF). The specificity of the ␣ 1E antisense oligonucleotides was supported by the lack of change in estimates of the P͞Q current amplitude. Furthermore, antisense and sense oligonucleotides against ␣ 1A did not affect R-type current expression (؊11.5 ؎ 1.7 and ؊11.7 ؎ 1.7 pA͞pF, respectively), whereas the ␣ 1A antisense oligonucleotide significantly reduced whole cell currents under conditions in which P͞Q current is dominant. Our results support the hypothesis that members of the E class of ␣ 1 subunits support the high voltage-activated R-type current in cerebellar granule cells.Individual nerve cells in the vertebrate nervous system express several types of voltage-gated Ca 2ϩ channel (1-4), as many as five or six channel types distinguishable in some neurons (5). These channels work together to support fundamental cellular activities such as membrane excitation, neurotransmitter release, neurite outgrowth, and gene expression (6, 7). Considerable advances have been made in the understanding of the relationship between channel types, defined by their biophysical and pharmacological characteristics, and their underlying ␣ 1 subunits isolated by biochemistry and molecular biology (8)(9)(10)(11)(12). It is clear that L-type currents are supported by dihydropyridine-sensitive ␣ 1C or ␣ 1D subunits (13,14) and N-type currents are generated by -conotoxin-GVIA-sensitive ␣ 1B subunits (15). Likewise, P-and Q-type currents are likely to arise from -Aga-IVA and -CTx-MVIIC-sensitive ␣ 1A subunits (16-21).Among the major categories of Ca 2ϩ channels uncovered so far, R-type channels were the most recently defined and remain the least well-understood. R-type currents were first identified in rat cerebellar granule neurons (22, 23) and were found to be pharmacologically and kinetically distinguishable from L-, N-, P-, and Q-type currents in the same cells (5). The importance of R-type channels for dendritic Ca 2ϩ entr...

T-type current modulation by the actin-binding protein Kelch-like 1

“…Cerebellar granule neurons (CGN) and hippocampal neurons were cultured as previously reported (32,33). In brief, one cerebellum from a 5-to 7-day-old mouse (or 2 hippocampi from a 0 -1 day old) was removed and washed in modified Hanks' balanced salt solution (HBSS) (Sigma, St. Louis, MO).…”

Signaling by Bone Morphogenetic Proteins and Smad1 Modulates the Postnatal Differentiation of Cerebellar Cells

“…To enhance uptake into the cells, three bases at the ends of the ODNs were modified to contain phosphorothioate bonds (Pilowsky et al, 1994). Previous studies have demonstrated that the treatment of cerebellar cells in culture with these modified ODNs reduces the levels of targeted proteins without altering cell viability (Piedras-Renteria and Tsien, 1998;Kumar et al, 2001). The probes had the following sequences: Smad1 ODN, 5Ј-TGAAAACAAGTGGTCA-3Ј; and SMAD4 ODN, 5Ј-CTGTGGCACATCAAACT-3Ј.…”

Reduction of insulin secretion in the insulinoma cell line INS-1 by overexpression of a Ca(v)2.3 (alpha1E) calcium channel antisense cassette

“…Figure 2 Detection of sense and antisense a1E cassettes by RT-PCR in stably transfected INS-1 cell clones. The full-length cDNA of sense and antisense cassettes was amplified after reverse transcription (see lanes with '+') of total RNA from four antisense (lanes 2-9) and two sense clones (lanes [11][12][13][14]. Temperature and cycle number were selected to obtain optimal conditions for a selective amplification of only overexpressed transcripts.…”

“…Using antisense a1E oligonucleotides and the a1E-selective antagonist SNX-482, it was shown that a1E is related to multiple resistant components of HVA Ca 2+ channels in rat cerebellar granule cells (12,13). Some, but not all of the resistant component is absent in a1E-depleted cerebellar granule cells isolated from a1E knockout mice (14).…”