Antisense Oligonucleotides against α 1E Reduce R-Type Calcium Currents in Cerebellar Granule Cells

Many neurons of the central nervous system display multiple high voltage-activated Ca²⁺ 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 α_1E subunit, which yields Ca²⁺ currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against α_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 α_1E antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from -10 ± 0.6 picoamperes/picofarad (pA/pF) (n = 6) in the untreated controls to -4.8 ± 0.8 pA/pF (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 pA/pF). The specificity of the α_1E antisense oligonucleotides was supported by the lack of change in estimates of the P/Q current amplitude. Furthermore, antisense and sense oligonucleotides against α_1A did not affect R-type current expression (-11.5 ± 1.7 and -11.7 ± 1.7 pA/pF, respectively), whereas the α_1A antisense oligonucleotide significantly reduced whole cell currents under conditions in which P/Q current is dominant. Our results support the hypothesis that members of the E class of α₁ subunits support the high voltage-activated R-type current in cerebellar granule cells.