A novel nervous system beta subunit that downregulates human large conductance calcium-dependent potassium channels

Abstract

The pore-forming alpha subunits of many ion channels are associated with auxiliary subunits that influence channel expression, targeting, and function. Several different auxiliary (beta) subunits for large conductance calcium-dependent potassium channels of the Slowpoke family have been reported, but none of these beta subunits is expressed extensively in the nervous system. We describe here the cloning and functional characterization of a novel Slowpoke beta4 auxiliary subunit in human and mouse, which exhibits only limited sequence homology with other beta subunits. This beta4 subunit coimmunoprecipitates with human and mouse Slowpoke. beta4 is expressed highly in human and monkey brain in a pattern that overlaps strikingly with Slowpoke alpha subunit, but in contrast to other Slowpoke beta subunits, it is expressed little (if at all) outside the nervous system. Also in contrast to other beta subunits, beta4 downregulates Slowpoke channel activity by shifting its activation range to more depolarized voltages and slowing its activation kinetics. beta4 may be important for the critical roles played by Slowpoke channels in the regulation of neuronal excitability and neurotransmitter release.

Fig. 1.

Sequence analysis of Slowpoke β subunits.a, Amino acid sequences of hβ4 (GenBank accession number AF215891), mβ4 (GenBank accession number AF215892), and hβ1 (GenBank accession number U38907) are aligned by the clustal method. Amino acids conserved in the β subunits are boxed. Thehorizontal bars indicate the two predicted membrane-spanning regions in β4, and conserved cysteine residues in the predicted extracellular loops are marked by arrows.b, Phylogenetic tree of Slowpoke β subunits cloned to date. The length of each pair of branches represents the evolutionary distance between sequence pairs, as measured by the number of substitution events. The Slowpoke β4 subunits described in this paper form a gene family distinct from other β subunits, which fall into a separate and evolutionarily conserved family. The GenBank accession numbers for the previously cloned β subunits used in this analysis are as follows: rat β, 1718491; dog β, 1127826; cow β, 508846; rabbit β, 2662318; hβ1, U38907; mβ1, 2347044; quail β, U67865; and hβ2/3, AF099137.

Fig. 2.

Analysis of β4 expression using human multiple tissue (top panels) and brain region (bottom panels) Northern blots. A predominant 1.6 kb band is detected in brain, particularly in cortical regions, and a minor 5 kb band is also seen in brain. Fainter signals can also be detected in peripheral tissues. Lanes: 1, heart; 2, brain;3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney,8, pancreas; 9, spleen;10, thymus; 11, prostate;12, testes; 13, ovary; 14, small intestine; 15, colon; 16, peripheral blood leukocyte; 17, cerebellum;18, cerebral cortex; 19, medulla;20, spinal cord; 21, occipital lobe;22, frontal lobe; 23, temporal lobe;24, putamen; 25, amygdala;26, caudate nucleus; 27, corpus callosum;28, hippocampus; 29, whole brain;30, substantia nigra; 31, subthalamic nuclei; 32, thalamus.

Fig. 3.

Expression of β4 in human cortex. Emulsion autoradiograms of sections from human cortex, labeled with human β4 antisense (β4 AS, left panel) and sense (β4 S,middle panel) probes, were viewed under dark-field illumination (10× magnification). The antisense probe shows β4 expression throughout the cortical layers, whereas the sense probe shows only background. A bright-field image of the same tissue (β4 AS, right panel) shows β4 expression (arrowheads) in cortical neurons (40× magnification).

Fig. 4.

Expression of Slowpoke α and β subunits in monkey brain. Film autoradiograms of monkey brain sections hybridized with antisense probes that detect mRNA encoding Slowpoke α subunit (Slo), β4, β2/3, and β1. Note the overlapping expression of α subunit (Slo) and β4 in multiple brain regions, with considerably lower levels of β2/3 and little or no expression of β1.

Fig. 5.

Analysis of Slowpoke α and β subunit expression in different regions of monkey brain. Emulsion autoradiograms of monkey brain sections, labeled with antisense probes to detect Slowpoke α (Slo, top row), β4 (second row), β2/3 (third row), and β1 (bottom row) subunits, were viewed under dark-field illumination. Slo and β4 are expressed in cortex (CTX), in the dentate gyrus (arrow) and CA3 (arrowhead) regions of hippocampus (HIP), and in thalamus (THL). β2/3 is expressed at lower levels and in apparently fewer cells in cortex and hippocampus and is not detected in thalamus. β1 expression is not detectable in any of these areas (10× magnification).

Fig. 6.

Analysis of Slowpoke α and β subunit expression in monkey aorta. Emulsion autoradiograms of sections of monkey aorta labeled with antisense probes to Slowpoke α (Slo, top panel), β4 (middle panel), and β1 (bottom panel) subunits were viewed under dark-field illumination. Silver grains representing mRNA encoding α and β1 subunits are readily visible over smooth muscle layers in the wall of the monkey aorta, whereas β4 expression is not detectable (10× magnification).

Fig. 7.

Mammalian Slowpoke α subunits coimmunoprecipitate with β4 subunits. Shown are Western blots of cell lysates or IPs, using an antibody directed against the V5 epitope that detects V5-His tagged β4 subunits. a, Human proteins. Lane 1, hSlo IP from cells transfected with hSlo and hβ4; lane 2, lysate from same cells as in lane1; lane3, anti-His IP from same cells as in lane 1; lane 4, hSlo IP from cells transfected with hSlo alone; lane 5, hSlo IP from cells transfected with hβ4 alone. b, Mouse proteins.Lane 1, mSlo IP from cells transfected with mSlo and mβ4; lane 2, lysate from same cells as in lane 1; lane3, anti-His IP from same cells as inlane 1.

Fig. 8.

hβ4 decreases Slowpoke activation rate but does not affect its deactivation rate. a, Normalized currents recorded at +80 mV from cells transfected with either hSlo and control vector, or hSlo and hβ4. b and c show time constants (τ) calculated from single exponential fits of current traces. b, Activation kinetics: hβ4 increases the activation time constant significantly (p < 0.001), whereas hβ1 is without effect (p> 0.05). c, Deactivation kinetics: hβ4 does not alter the deactivation time constant (p > 0.05), whereas hβ1 increases it significantly (p< 0.001). Recording conditions: detached inside-out patches, symmetrical K⁺ solutions, 1 μm free Ca²⁺ on the intracellular side, holding potential of −100 mV, steps to +80 mV.

Fig. 9.

Steady-state activation of hSlo in the presence or absence of hβ1 or hβ4. a, hβ4 shifts the steady-state activation curve to the right, indicating that more depolarized voltages are required to open the channel.V₅₀ (indicated by the dotted lines) is the half maximal activation voltage for each condition. Holding potential of −100 mV, 1 μm free calcium on the intracellular side, data fitted to the Boltzmann equation (n = 11 for hSlo plus vector;n = 10 for hSlo plus hβ4). b,V₅₀ is shifted in the depolarizing direction by hβ4 at all calcium concentrations tested. In contrast, hβ1 shifts the half-maximal activation voltage in the hyperpolarizing direction.

Fig. 10.

hβ4 protects hSlo from block by iberiotoxin (a) and charybdotoxin (b). Experiments were done in the whole-cell patch-clamp mode, and toxins were applied from the extracellular side. hSlo currents recorded in the presence of different concentrations of toxins were normalized to the control current in the absence of toxin. The filled symbols with solid lines show the block of current by toxins in the absence of hβ4, and the open symbols with dashed lines show that current is not blocked, even by high concentrations of toxin, in the presence of hβ4 (n = 3; holding potential of −85 mV, steps to +105 mV, 10 μm free internal calcium). Note the different concentration axes in a andb.