The expression of two splice variants of the Kv3.1 potassium channel gene is regulated by different signaling pathways

Abstract

The Kv3.1 potassium channel gene gives rise to two different channel proteins, Kv3.1a and Kv3.1b, by alternative splicing of nuclear RNA. During development the levels of Kv3.1b mRNA (but not Kv3.1a) substantially increase in rat cerebellum after postnatal day 8. The molecular mechanism underlying the differential regulation of the two transcripts is not known. Using in vitro slices of cerebellum, we have found that basic fibroblast growth factor (bFGF) upregulates both Kv3.1a and Kv3.1b at this developmental stage, but that depolarization by elevated potassium concentrations is without effect. Combined treatment with bFGF and depolarization, however, prevents the increase in Kv3.1a transcripts and selectively increases Kv3.1b mRNA levels. A protein kinase C (PKC) inhibitor blocks the increase in Kv3.1a mRNA levels induced by bFGF alone but does not affect the increase in Kv3.1b mRNA. Measurement of nuclear protein kinase C activity shows that bFGF activates this enzyme and that depolarization blocks this activation. In contrast to these findings at postnatal day 8, bFGF fails to alter Kv3.1 transcripts in slices from adult animals, and PKC activity is enhanced rather than suppressed by depolarization. Our results indicate that different signaling pathways regulate Kv3.1a and Kv3.1b expression and suggest that Kv3.1a mRNA levels may be modulated by neuronal activity.

Fig. 1.

Expression levels of Kv3.1a and Kv3.1b mRNA during development. A, RNase protection analysis of Kv3.1a and Kv3.1b mRNA. The protected bands at 398, 316, and 108 nucleotides correspond to Kv3.1b, GAPDH, and Kv3.1a mRNAs, respectively. Thebottom panel shows the Kv3.1 band visualized after a longer exposure to film. B, Densitometric measurements of the relative amounts of the Kv3.1a and Kv3.1b mRNAs. The intensities of Kv3.1a bands were multiplied by 3.6 to account for the differences in the number of labeled C residues in the protected bands of Kv3.1a and Kv3.1b, as described previously (Perney et al., 1992). The levels of the Kv3.1a and Kv3.1b mRNA are normalized to the total amount of GAPDH mRNA. Each point is the mean of two measurements from a single experiment.

Fig. 2.

Modulation of Kv3.1 mRNA levels by bFGF during development. A, Effects of bFGF on the levels of the Kv3.1a and Kv3.1b mRNA detected by an RNase protection assay at postnatal days 8 and 15. Total RNA was isolated from cerebellums immediately after slice preparation from cerebellar slices incubated in ACSF and from slices treated with 100 ng/ml bFGF in ACSF for 6 hr at room temperature. The bands corresponding to Kv3.1b, GAPDH, and Kv3.1a mRNAs are 398, 316, and 108 nucleotides, respectively.B, Summary of the effects of bFGF on the Kv3.1a and Kv3.1b mRNA levels at P3, P8, and P15. All values are mean ± SEM. The changes in Kv3.1a and Kv3.1b mRNA levels at P8 are significantly different from 0; p < 0.01 andp < 0.05, respectively. The changes in Kv3.1a and Kv3.1b mRNA levels are also significantly different by ANOVA testing among these age groups, with p < 0.05. A Tukey–Kramer multiple-comparisons test showed that the change in Kv3.1b mRNA expression at P8 was significantly different from that at P3, and that the change in the Kv3.1a mRNA levels at P8 was significantly different from that at P3 and P15; p< 0.05. The n values for Kv3.1a were 5 at P3, 4 at P8, and 3 at P15. The n values for Kv3.1b at P3, P8, and P15 were 5, 5, and 3, respectively.

Fig. 3.

Differential regulation of Kv3.1a and Kv3.1b mRNA at P8 by bFGF and high-K ACSF. A, RNase protection analysis of effects of depolarization on the FGF-induced upregulation of Kv3.1a mRNA levels. Cerebellar slices were treated with 100 ng/ml bFGF in ACSF or with bFGF in high-K ACSF. The bands corresponding to the Kv3.1b, GAPDH, and Kv3.1a mRNAs are 398, 316, and 108 nucleotides, respectively. B, C, Summary of effects of depolarization on the FGF-induced changes in the levels of Kv3.1 transcripts. Cerebellar slices were treated with bFGF in ACSF, bFGF in high-K ACSF, or with high-K ACSF alone for 6 hr (B), or with bFGF in ACSF for 1 hr followed by incubation in ACSF for 5 hr (C). The interaction between FGF and high K treatment is significant (p < 0.05) for Kv3.1a by a two-factor ANOVA test (n = 4). The change induced by FGF is significantly different from that in control and from that in high-K plus FGF-treated sample (p < 0.01), using the Tukey’s multiple-comparisons test. The change in the Kv3.1a mRNA levels induced by a 6 hr bFGF treatment (n = 4) is different from that induced by a 1 hr bFGF treatment followed by 5 hr incubation in ACSF (n = 3); p< 0.05, by a one-tailed Student’s t test.

Fig. 4.

Effects of kinase inhibitors and an inhibitor of protein synthesis on the FGF-induced upregulation of Kv3.1 mRNA in P8 cerebellum. Slices were treated with 100 ng/ml bFGF in the presence of 50 μm AFC (an inhibitor of ras), 10 μm KN-62 (CaM/kinase inhibitor), 10 μmKT5720 (PKA inhibitor), 2.5 μm BIM I (PKC inhibitor), and 5 μg/ml cycloheximide in ACSF for 6 hr. The change in Kv3.1a mRNA levels induced by bFGF is significantly different from the changes produced by bFGF in the presence of KT5720, KN-62, AFC, BIM, and cycloheximide, with p < 0.05, 0.05, 0.005, 0.005, and 0.001, respectively, using two-tailed Student’s ttests (n = 3). The FGF-induced change in Kv3.1b mRNA levels is significantly different from that produced by FGF plus cycloheximide; p < 0.05 (n = 3).

Fig. 5.

Inhibition of FGF-induced nuclear PKC activation by high-K ACSF. Cerebellar slices were incubated in ACSF alone, in ACSF containing 100 ng/ml bFGF, in bFGF plus high-K ACSF, or in bFGF plus 2.5 μm PKC inhibitor BIM I at room temperature for 2 hr. Nuclei were isolated, and nuclear PKC activity was determined immediately. The nuclear PKC activity from FGF-treated cells differs significantly (p < 0.05) from that in control, bFGF plus high K, and bFGF plus BIM samples using a one-tailed Student’s t test (n = 3).

Fig. 6.

The effects of high-K ACSF treatment on PKC activity of P8 (A) and adult (B) cerebellum. P8 and adult cerebellar slices were incubated in ACSF or high-K ACSF for 2 hr. PKC activity of homogenates was determined in the presence or absence of 10 μm PMA. Changes in PKC activity were calculated according to the following equation: (PKC activity (high K)/PKC activity (ACSF) −1) × 100%. The PMA-independent PKC activity in control (ACSF) sample at P8 is significantly different from that in the high-K treated sample; p < 0.05, by a one-tailed Student’st test. The interaction between age of the animal and the effects of high K treatment is significant; p< 0.05 for PMA-independent PKC activity; p < 0.01 for the PKC activity in the presence of PMA, using a two-factor ANOVA test (n = 4). The PKC activity in adult high-K-treated sample is significantly different from that in the adult control (ACSF) and in the P8 high-K-treated sample;p < 0.01 (without PMA) and p< 0.01 (with PMA). PKC activity in the adult control is different from that in P8 control; p < 0.05 (without PMA) andp < 0.01 (with PMA), by a Tukey’s multiple-comparisons test. The depolarization-induced change in PKC activity in the absence and presence of PMA in the adult is significantly different from that at P8; p < 0.001 (without PMA) and p < 0.01 (with PMA), by a two-tailed Student’s t test.

Fig. 7.

The inhibitory effects of P8 homogenates on PKC activity. PKC activity of P8 and adult cerebellar homogenates, and of homogenates of P8 and adult cerebellar slices that were preincubated in ACSF or high-K ACSF, was determined individually. The homogenates of two samples (a, b), indicated on thex-axis, were then mixed, and the PKC activity of the combined sample (a, b) was measured. The PKC activity in the combined sample and the sum of the PKC activity of the two samples is shown in A (without PMA) andB (with 10 μm PMA). The sum of the PKC activity in the adult (high K) sample and in P8, P8 (ACSF), P8 (high K) samples is significantly different from that in the combined samples, adult (high K) plus P8, adult (high K) plus P8 (ACSF), and adult (high K) plus P8 (high K), with p < 0.05, 0.01, and 0.002 (without PMA), respectively, and p < 0.01, 0.05, and 0.02 (with PMA), respectively (n = 3), by a two-tailed Student’s t test. The PKC activity in the combined sample, P8 plus P8 (high K), is also different from the sum of PKC activity in the P8 and in P8 (high K) samples;p < 0.05 (without PMA) and p< 0.01 (with PMA) (n = 3), by a two-tailed Student’s t test. Changes in PKC activity resulting from the mixing of the two samples (C) were calculated, using the equation [(a +b/(a + b)) − 1] × 100%.