Regional differences in distribution and functional expression of small-conductance Ca2+-activated K+ channels in rat brain

Abstract

Small-conductance Ca2+-activated K+ (SK) channels are important for excitability control and afterhyperpolarizations in vertebrate neurons and have been implicated in regulation of the functional state of the forebrain. We have examined the distribution, functional expression, and subunit composition of SK channels in rat brain. Immunoprecipitation detected solely homotetrameric SK2 and SK3 channels in native tissue and their constitutive association with calmodulin. Immunohistochemistry revealed a restricted distribution of SK1 and SK2 protein with highest densities in subregions of the hippocampus and neocortex. In contrast, SK3 protein was distributed more diffusely in these brain regions and predominantly expressed in phylogenetically older brain regions. Whole-cell recording showed a sharp segregation of apamin-sensitive SK current within the hippocampal formation, in agreement with the SK2 distribution, suggesting that SK2 homotetramers underlie the apamin-sensitive medium afterhyperpolarizations in rat hippocampus.

Fig. 1.

Immunological identification of SK channel protein in rat hippocampus and Xenopus oocyte membranes: characterization of anti-SK antibodies in Western blotting experiments. Twenty micrograms of purified rat hippocampal synaptic plasma membrane vesicles or Xenopus oocyte membranes expressing SK1, SK2, or SK3 channels were separated by 10% SDS-PAGE and transferred to PVDF membranes. SK proteins were detected by the respective antibodies following standard procedures. SK1 protein, 65, 58, and 43 kDa in rat brain and 69 kDa in oocyte membranes (N-terminal AB); SK1 protein, 65 and 58 kDa in rat brain membranes (C-terminal AB); SK2 protein, 67 kDa in rat brain and 64 kDa in oocyte membranes; SK3 protein 70 kDa in rat brain and 67 kDa in oocyte membranes. AB, Antibody; I, immune serum;PI, preimmune serum; RB, rat brain membranes; OO, oocyte membranes;

Fig. 2.

Immunoprecipitation of detergent-solubilized SK channel complexes. A, Cholate-solubilized SK channels were immunoprecipitated using increasing concentrations of anti-SK1_(12–29) (▴), anti-SK2_(538–555)(▪), or anti-SK3_(504–522) (●) antibodies immobilized on protein A-Sepharose. Immunoprecipitated channels were quantified by [¹²⁵I]apamin binding to antibody-bound SK channels. In this experiment, anti-SK2_(538–555) saturably precipitated 2100 cpm, whereas anti-SK3_(504–522)immobilized 930 cpm. Anti-SK1_(12–29) was unable to precipitate any [¹²⁵I]apamin binding. One representative experiment is shown. PI, Preimmune serum.B, Cholate-solubilized neuronal SK channels were immunoprecipitated by a saturating amount of anti-SK2_(538–555) and anti-SK3_(504–522)antibodies or a combination thereof. The extent of immunoprecipitation was quantified by [¹²⁵I]apamin binding to antibody-immobilized SK channels. Exclusively anti-SK2_(538–555) and anti-SK3_(504–522)antibodies were capable of immunoprecipitating [¹²⁵I]apamin binding, whereas the corresponding preimmune sera did not immobilize soluble apamin-sensitive SK channels.C, Solubilized neuronal SK channels were immunoaffinity-purified by column chromatography using anti-SK2_(538–555) or anti-SK3_(504–522). Eluates were analyzed by Western blotting using anti-SK antibodies as indicated. Note that the SK3 antibody does not recognize anti-SK2-immunoprecipitated material and vice versa. D, Immunoblot analysis of anti-SK2_(538–555)- and anti-SK3_(504–522)-immunoprecipitated material for the presence of calmodulin. Equal amounts of detergent-solubilized material were subjected to immunoprecipitation. Immunoreactivity observed in thecontrol lane corresponds to monomeric and dimeric calmodulin.

Fig. 3.

Coronal sections showing the distribution of SK1–SK3 protein in rat brain. Low-magnification microphotographs show the overall distribution of SK1–SK3 immunoreactivity in coronal brain sections. Adjacent sections (40 μm) were stained with affinity-purified anti-SK1_(12–29) (2.0 ng/μl IgG), anti-SK2_(538–555) 1:6000 (crude serum), and anti-SK3_(504–522) (1.3 ng/μl IgG). Nonspecific immunostaining was assessed using preimmune serum. Immune serum preadsorbed to immunogenic peptide yielded essentially identical results. 3V, Third ventricle; Amyg, amygdala, CM, centromedial thalamic nucleus;CPu, caudate putamen; Cx, neocortex;Hi, hippocampus; LD, laterodorsal thalamic nucleus; LH, lateral hypothalamic area;PV, paraventricular thalamic nucleus; Rt, reticular thalamic nucleus; VP, ventroparietal thalamic nucleus. Scale bar, 1000 μm.

Fig. 4.

Differential distribution of SK1–SK3 protein in sections of the rat brain: expression of SK proteins in subsets of neurons. A–C, SK1–SK3-IR in layers IV–VI of the frontoparietal cortex (coronal brain section). D–F, SK1–SK3 localization in the CA3 region of the hippocampus.G–I, Localization of SK1–SK3 protein within the hippocampus proper. J–L, Distribution of SK1–SK3 protein within the dentate gyrus. gl, Granule cell layer; ml, molecular layer; pl, polymorphic layer; sl, stratum lucidum;sp, stratum pyramidale; so, stratum oriens; sr, stratum radiatum. Scale bar:A–C, 45 μm; D–F, J–L, 100 μm;G–I, 400 μm.

Fig. 5.

Apamin-sensitive and -insensitive AHP currents in hippocampal pyramidal cells and DG cells. Typical AHP currents recorded in a CA1 pyramidal cell (A), a CA3 pyramidal cell (B), and a DG granule cell (C) in rat hippocampal slices are shown. Each cell was voltage-clamped at –55 mV in the presence of TTX and TEA in the extracellular medium to suppress the Na⁺- and BK-channel-mediated currents. The AHP currents were elicited by a brief depolarizing voltage step (100 msec to 0 mV) once every 60 sec. The depolarizing step elicited biphasic outward tail currents in all three cell types (Control): an early tail current of medium duration was followed by an I_sAHPlasting several seconds. The early current was much larger in CA1 and CA3 pyramidal cells than in DG granule cells. Bath application of 100 nm apamin abolished the early outward tail current in the CA1 and CA3 pyramidal cells but had little or no effect in the DG cells. The records before and after apamin application are compared (Superimposed) at two different time scales to show the time course of both components. (The current records in the CA3 cell appeared “noisy” because of a large number of spontaneous miniature synaptic currents.) D–G, Summary data comparing currents in CA1 pyramidal cells (n = 6), CA3 pyramidal cells (n = 9), and DG granule cells (n = 6). D, Peak amplitudes of the apamin-sensitive tail current obtained by digital subtraction of records taken before and after application of 100 nmapamin. The current was measured 50–60 msec after the end of the depolarizing step. E, Peak amplitudes of the apamin-insensitive sAHP current. F, G, Current densities obtained by dividing the measurements shown inD and E, respectively, by the whole-cell capacitance measured in each cell. F, Current densities for the apamin-sensitive tail current. G, Current densities for the apamin-insensitive sAHP current. All currents (A–G) were recorded without cAMP in the pipette and in extracellular medium with 1 μm TTX and 5 mm TEA. D–G, Mean values and SEM (error bars) for the peak tail currents.

Fig. 6.

Apamin-sensitive currents in CA1 and CA3 pyramidal cells and DG granule cells recorded after suppression of the sAHP BK- and M-currents. To study the apamin-sensitive currents in relative isolation, the I_sAHP BK current and M-current were suppressed by including 100 μm cAMP (cAMP or 8CPT-cAMP) in the intracellular medium of the recording pipette and 5 mm TEA plus 10 μmXE991 in the extracellular medium throughout each experiment. Tail currents from representative CA1 (A) and CA3 (B) pyramidal cells and a DG granule cell (C) are shown. The currents were elicited and recorded using the same voltage-clamp protocol as in Figure 5 (holding potential, –55 mV; 100 msec step). For each cell (A–C), records before (Control) and after application of 100 nm apamin are shown. Note that the DG granule cells showed no net outward tail current under these conditions. Furthermore, the apamin-sensitive current isolated by digital subtraction (Subtracted) was far smaller in the DG neurons than in the CA1 and CA3 neurons and decayed more slowly in CA3 than in CA1 cells. Superimposed, Comparison of the records before and after apamin application. D, E, Comparison between typical time courses of the effect of apamin (100 nm) application (gray bar) on the early tail current (normalized; control = 100%) in a pyramidal cell (D) and a DG granule cell (E). The apamin effects in CA1 and CA3 pyramidal cells were similar (the example shown in D is from a CA3 cell), whereas the effect was much weaker in DG cells. Note in both cell types the slight time-dependent run-down of the currents. In pyramidal cells, an initial increase (“run-up”) of the current amplitude was sometimes observed (D; 0–3 min).F, G, Summary data comparing apamin-sensitive currents (F) and current densities (G) in CA1 pyramidal cells (n= 9), CA3 pyramidal cells (n = 8), and DG granule cells (n = 7). D, Peak amplitudes of the apamin-sensitive tail current obtained by subtraction of records before and after application of 100 nm apamin.G, Current densities obtained by dividing the measurements shown in F by the whole-cell capacitance measured in each cell.