hSK4, a member of a novel subfamily of calcium-activated potassium channels

Abstract

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.

Figure 1

Primary structure and tissue distribution of hSK4. (a) Amino acid sequence alignment of hSK4 and rSK1 with hSK1, rSK2, and rSK3. Sequences were aligned with the computer program pileup (GCG) using default parameters. Gaps are represented by dots. A dark line is drawn above putative transmembrane domains, denoted by the labels S1-S6, in addition to the P region. Shading denotes absolutely conserved residues. Consensus sites for phosphorylation by protein kinases A and C are marked by open squares and circles, respectively. Leucine heptad repeats are indicated by darkened boxes. The National Center for Biotechnology Information accession numbers for the nucleotide sequences of hSK4 and rSK1 are AF000972 and AF000973, respectively. (b) Kyte–Doolittle hydrophilicity analysis of hSK4 using a window of 20 residues. Numbers along the vertical axis refer to free energy of transfer to water. (c) Dendrogram based on the alignment in a. Horizontal branch lengths are inversely proportional to the similarity between sequences, whereas vertical branch lengths are for illustrative purposes only. (d) Northern blot analysis of hSK4 transcript using 3′ untranslated cDNA as probe. Molecular sizes are indicated in kilobases.

Figure 2

hSK4 generates a Ca²⁺-dependent, K⁺-selective current. (a) Whole-cell currents from a single cell patched sequentially with 0 (Top), with 907 nM (Middle), and again with 0 free Ca²⁺ (Bottom) in the pipette. From a holding potential of −70 mV, the membrane was given 200-ms test pulses from −140 to 80 mV in 20-mV increments. Similar results were obtained in 4/4 cells tested. (b) Current-voltage relation for the same cell as in a. (c) Sensitivity of hSK4 current to Ca²⁺. Whole-cell currents were measured from cells with 29, 91, 288, or 907 nM free Ca²⁺ in the pipette. The current density of a given cell was calculated by dividing current amplitude at 80 mV by cell capacitance. The average current density of five different cells at each Ca²⁺ concentration is shown. Fitting these data to the Hill equation yielded an EC₅₀ of 95 nM Ca²⁺ and a Hill coefficient of 3.2. (d) Extracellular K⁺ shifted the membrane potential as predicted by the Nernst equation for a K⁺-selective channel. The membrane potential was recorded with 907 nM free Ca²⁺ in the pipette as bath KCl was changed from 3 to 33 mM. Each point represents the average of six cells. A linear regression through these points yields a slope of 56 mV per 10-fold change in bath K⁺ concentration.

Figure 3

A representative inside-out patch perfused sequentially with 0, 29 nM, and 91 nM free Ca²⁺ activates increasing numbers of hSK4 channels. Washout with 0 Ca²⁺ reverses the effect. In this and in identical experiments with other cells, patches were held at 35 mV (intracellular positive) with 130 mM K⁺ in the bath and 3 mM K⁺ in the pipette.

Figure 4

Single-channel recordings indicate hSK4 has a small conductance. (a) Discrete single-channel activity recorded continuously from a representative inside-out patch at 35 mV (intracellular positive) in the presence of 29 nM free Ca²⁺ in the bath. Occasional transitions to two and three openings indicate that multiple channels are present in this patch. (b) An all-points histogram of the recording in a yields two distinct peaks at 1.5 pA and 2.9 pA. (c) Single-channel current-voltage relation for the same patch, using data acquired as in b at 0, 10, and 35 mV. With 130 mM K⁺ in the bath and 3 mM K⁺ in the pipette, linear regression yields a reversal potential of −96 mV and a slope conductance of 12 pS.

Figure 5

hSK4 currents are blocked strongly by extracellular Ba²⁺ and charybdotoxin, weakly by TEA, and not at all by apamin or by Cs⁺. (a) Currents after drug treatment normalized to pretreatment levels. From a holding potential of −80 mV and with 907 nM free Ca²⁺ in the pipette, whole-cell currents were measured during 100–150-ms test pulses to 80 mV before and after extracellular application of 1 mM BaCl₂ (Ba²⁺), 20 nM charybdotoxin (CTX), 5 mM TEA, 100 nM apamin (APA), or 1 mM CsCl (Cs⁺). For all recordings 130 mM K⁺ was present in the pipette, and 3 mM K⁺ was present in the bath. Ba²⁺ reduced hSK4 currents by 88 ± 3% (n = 5, P < .0005 by paired t test) and had an IC₅₀ of 340 μM (data not shown). Charybdotoxin reduced hSK4 currents by 88 ± 4% (n = 6, P < .005 by paired t test) and had an IC₅₀ of 2.0 nM (data not shown). TEA reduced hSK4 currents by 17 ± 3% (n = 5, P < .005 by paired t test). Apamin and Cs⁺ had no effect at the concentrations used here. (b) A representative recording of hSK4 currents before and after bath application of 1 mM BaCl₂. In addition to blocking hSK4 currents, Ba²⁺ appears to alter hSK4 kinetics. (c) A representative recording of hSK4 currents before and after bath application of 20 nM charybdotoxin. Block by toxin appears to be relieved over the course of depolarizing pulses.