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Small-Conductance, Calcium-Activated Potassium Channels from Mammalian Brain

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Science  20 Sep 1996:
Vol. 273, Issue 5282, pp. 1709-1714
DOI: 10.1126/science.273.5282.1709

Figures

  • Fig. 1.

    (A) Amino acid sequences (19) predicted for hSK1, rSK2, rSK3, and the partial clone, rSK1. Alignments were generated by eye; dots represent gaps introduced to optimize the alignment. The six predicted transmembrane domains and the pore region are overlined. Residues that are conserved among all of the clones are boxed. Amino acid numbers for the full-length coding sequences are given on the right. The GenBank accession numbers for these sequences are as follows: rSK2, U69882; hSK1, U69883; rSK3, U69884; and rSK1, U69885. The asterisks indicate stop codons. (B) Hydropathy plot for rSK2 calculated from the algorithm of Kyte and Doolittle with an amino acid window of nine residues. HPhobic, hydrophobic; HPhilic, hydrophilic. (C) Dendrogram of representative mammalian members of the K+ channel superfamily (18). The horizontal branch length is inversely proportional to the similarity between sequences.

  • Fig. 2.

    In situ hybridization and Northern blot analysis for rSK mRNAs. (A) Autoradiogram of in situ hybridization with an antisense rSK1 RNA probe. Hybridization was found in the hippocampus (CA3), the dentate gyrus (DG), the subiculum (S), the anterior olfactory nucleus (AON) and olfactory tubercle (Tu), the cerebellum (Cb), and the cortex (Ctx). (B) Autoradiogram of in situ hybridization with an antisense rSK2 RNA probe. Of the SK mRNAs, rSK2 mRNA was the most widely expressed and was highest in the hippocampus (not labeled), with lower levels of expression in the olfactory bulb and the anterior olfactory nucleus, the granular layer of the cerebellum (not labeled), the reticular nucleus of the thalamus (Th), and the pontine nucleus (Pn). (C) Autoradiogram of in situ hybridization with an antisense rSK3 RNA probe. Hybridization was seen in the lateral septum and ventral tegmental area, the olfactory tubercle (not labeled), the caudate-putamen (Cp), the nucleus accumbens (Acb), the supraoptic nucleus (SO), many nuclei of the thalamus and hypothalamus (not labeled), and the substantia nigra pars compacta (SNC). Scale bar, 5 mm. (D) Northern blot analysis of rSK1 and rSK2 mRNAs. Polyadenylated [poly(A)+] mRNA (3 μg) from rat whole brain, heart, lung, spleen, adrenal gland, liver, kidney, and skeletal muscle was prepared as a Northern blot and probed with riboprobes specific for either rSK1 (right) or rSK2 (left); rSK1 mRNA was detected in rat brain (Br) and heart (He), and rSK2 mRNA was detected in brain and adrenal gland (Ad). Neither rSK1 nor rSK2 mRNA was detected from lung, liver, kidney, thymus, spleen, or skeletal muscle. Molecular sizes are indicated in kilobases.

  • Fig. 3.

    Expression of rSK2 and hSK1 in Xenopus oocytes. In (A) through (C), the metabotropic glutamate (Glut) receptor mGluR1a was expressed with or without rSK2 in Xenopus oocytes. Whole cell currents were measured from oocytes superfused with ND96 solution 2 to 3 days after mRNA injection. The holding potential was −80 mV. (A) Addition of glutamate (1 mM) to an oocyte injected with mGluR1a mRNA alone evoked a transient Ca2+-activated Cl current. Similar results were obtained in six other oocytes injected with mGluR1a. (B) Addition of glutamate (1 mM) to oocytes coinjected with mGluR1a and rSK2 mRNA evoked the transient Ca2+-activated Cl current observed with mGluR1a-injected oocytes, followed by a large transient outward current. Similar results were obtained in 14 other oocytes coinjected with mGlu1a and rSK2. (C) Injection of EGTA (final concentration, ∼10 mM) abolished the response to subsequent addition of glutamate in oocytes coinjected with mGluR1a and rSK2 mRNA. Similar results were obtained in three other oocytes coinjected with mGluR1a and rSK2. (D and E) Currents evoked by voltage steps from inside-out macropatches excised from an oocyte expressing rSK2 (D) or hSK1 (E). With 5 μM Ca2+ in the intracellular solution, the membrane was stepped from a holding potential of −80 mV to test potentials between −100 and 100 mV and then repolarized to −50 mV. Currents activated instantaneously and showed no inactivation during the 500-ms test pulses. (F and G) Current traces (I) elicited by 2.5-s voltage ramps (Vm) from −100 to 100 mV from inside-out macropatchs excised from oocytes expressing rSK2 (F) or hSK1 (G). The traces were obtained in the presence of the indicated concentrations of intracellular Ca2+; current amplitudes increased as the Ca2+ concentration was increased. (H and I) The relation between Ca2+ concentration and response obtained from the patches shown above for rSK2 (H) or hSK1 (I) channels. The slope conductance, G, at the reversal potential is plotted as a function of Ca2+ concentration. The data were fitted with the Hill equation, yielding a K0.5 of 0.43 μM and 0.71 μM and a Hill coefficient of 4.8 and 3.9 for rSK2 and hSK1, respectively.

  • Fig. 4.

    Single-channel recordings of rSK2. (A) Continuous recording at different internal Ca2+ concentrations from a representative inside-out patch containing rSK2 channels. Addition of 0.2 μM Ca2+ elicited openings to a single amplitude. Increasing Ca2+ to 0.4 μM increased channel activity, and openings to several levels are apparent. Upon addition of 0.6 μM Ca2+, channel activity increased such that discrete amplitudes could not be resolved. Channel activity ceased when Ca2+ was removed. Gaps represent breaks in the continuously acquired recording. The baseline (zero current level) of the segment recorded in 0.6 μM Ca2+ has been aligned by eye. For display, data were digitally filtered at 300 Hz. (B) Channel activity from a representative patch recorded in the presence of 0.4 μM Ca2+ at −60, −80, and −100 mV. The patch contained more than one channel, and double openings are apparent. For display, traces were digitally filtered at 300 Hz. (C) Single-channel current-voltage relation for the patch presented in (B). Data points were derived from the fitting of amplitude histograms at each membrane potential. Linear regression yielded a single-channel conductance of 10.8 pS, with a reversal at −6 mV.

  • Fig. 5.

    Pharmacology of rSK2 and hSK1. (A and C) Macroscopic rSK2 currents were recorded in 5 μM Ca2+ from inside-out macropatches with either 0 or 60 pM apamin (AP) (A) or 0 or 2 μM d-tubocurare (dTC) (C) in the extracellular solution. (B and D) Dose-response curves for block by external apamin [shown in (B) for rSK2] or d-tubocurare [shown in (D) for rSK2, closed circles, and hSK1, open circles]. Block was determined from multiple inside-out macropatches with or without drug. Each data point represents the fractional current (drug per control) at −100 mV from the average of six control patches and six subsequent patches with drug. Currents were elicited by voltage ramps as in (A). The continuous lines represent nonlinear least squares fits to the data for a 1:1 stoichiometry, giving a Ki of 63 pM for AP and 2.4 μM (rSK1) or 76.2 μM (hSK1) for dTC.

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