Chromaffin cells from mice lacking the BK β2 subunit show decreased action potential firing during current injection but an increase in spontaneous burst firing.

Ca2+- and voltage-gated K+ channels of large conductance (BK channels) are expressed in a diverse variety of both excitable and inexcitable cells, with functional properties presumably uniquely calibrated for the cells in which they are found. Although some diversity in BK channel function, localization, and regulation apparently arises from cell-specific alternative splice variants of the single pore–forming α subunit (KCa1.1, Kcnma1, Slo1) gene, two families of regulatory subunits, β and γ, define BK channels that span a diverse range of functional properties. We are just beginning to unravel the cell-specific, physiological roles served by BK channels of different subunit composition.

Two mammalian genes, Kcnt1 and Kcnt2, encode pore-forming subunits of Na+-dependent K+ (KNa) channels. Progress in understanding KNa channels has been hampered by the absence of specific tools and methods for rigorous KNa identification in native cells. Here, we report the genetic disruption of both Kcnt1 and Kcnt2, confirm the loss of Slo2.2 and Slo2.1 protein, respectively, in KO animals, and define tissues enriched in Slo2 expression. Noting the prevalence of Slo2.2 in dorsal root ganglion, we find that KO of Slo2.2, but not Slo2.1, results in enhanced itch and pain responses. In dissociated small diameter DRG neurons, KO of Slo2.2, but not Slo2.1, abolishes KNa current. Utilizing isolectin B4+ neurons, the absence of KNa current results in an increase in action potential (AP) firing and a decrease in AP threshold. Activation of KNa acts as a brake to initiation of the first depolarization-elicited AP with no discernible effect on afterhyperpolarizations.DOI: http://dx.doi.org/10.7554/eLife.10013.001

Many K+ channels are oligomeric complexes with intrinsic structural symmetry arising from the homo-tetrameric core of their pore-forming subunits. Allosteric regulation of tetramerically symmetric proteins, whether by intrinsic sensing domains or associated auxiliary subunits, often mirrors the fourfold structural symmetry. Here, through patch-clamp recordings of channel population ensembles and also single channels, we examine regulation of the Ca 2+ -and voltage-activated large conductance Ca 2+ -activated K + (BK) channel by associated γ1-subunits. Through expression of differing ratios of γ1:α-subunits, the results reveal an all-or-none functional regulation of BK channels by γ-subunits: channels either exhibit a full gating shift or no shift at all. Furthermore, the γ1-induced shift exhibits a state-dependent labile behavior that recapitulates the fully shifted or unshifted behavior. The γ1-induced shift contrasts markedly to the incremental shifts in BK gating produced by 1-4 β-subunits and adds a new layer of complexity to the mechanisms by which BK channel functional diversity is generated.-activated K + (BK) channels, like most K + channels and many other oligomeric protein complexes, exhibit structural symmetry that arises from the homo-tetrameric assembly of four identical pore-forming α-subunits (1-3). Structural symmetry in turn dictates many aspects of the functional behavior of such proteins. For BK channels, activation can occur either by voltage alone or independently by cytosolic Ca 2+ (4, 5). Activation initiated by voltage arises from independent movements of voltage sensors, one intrinsic to each α-subunit, which then allosterically couple to a conformational change, termed gating, that permits ion permeation through the central axis of the tetrad of subunits (5). For activation by Ca 2+ , up to two Ca 2+ binding events per α-subunit are thought to regulate the conformational status of a symmetric cytosolic gating ring (6-8), which then couples to pore opening. The mechanics and energetics of the gating process display a functional symmetry inherent from the independent participation of the sensing domains of each of the identical α-subunits (5). For BK channels, the pore-forming α-subunits can also associate with members of a family of four β-subunits (β1-β4) that define many tissue-specific properties of BK channels (9, 10), including apparent Ca 2+ dependence of activation (11, 12), inactivation (12-15), and pharmacology (12, 16). Insight into how such channel-associated proteins modify channel gating properties is therefore critical to understanding the physiological roles of ion channels in their native cellular environment. For BK channels, auxiliary β-subunits associate with α-subunits in an up to 1:1 stoichoimetry (Fig. S1A), maintaining the overall symmetry of the ion channel complex (3). However, functional channels can contain less than a full set of four β-subunits (3), contributing to the functional diversity of BK channels in native cells (17). In such cases each individual β-s...

Leucine-rich-repeat-containing protein 26 (LRRC26) is the regulatory γ1 subunit of Ca 2+ -and voltage-dependent BK-type K + channels. BK channels that contain LRRC26 subunits are active near normal resting potentials even without Ca 2+ , suggesting they play unique physiological roles, likely limited to very specific cell types and cellular functions. By using Lrrc26 KO mice with a β-gal reporter, Lrrc26 promoter activity is found in secretory epithelial cells, especially acinar epithelial cells in lacrimal and salivary glands, and also goblet and Paneth cells in intestine and colon, although absent from neurons. We establish the presence of LRRC26 protein in eight secretory tissues or tissues with significant secretory epithelium and show that LRRC26 protein coassembles with the pore-forming BK α-subunit in at least three tissues: lacrimal gland, parotid gland, and colon. In lacrimal, parotid, and submandibular gland acinar cells, LRRC26 KO shifts BK gating to be like α-subunit-only BK channels. Finally, LRRC26 KO mimics the effect of SLO1/BK KO in reducing [K + ] in saliva. LRRC26-containing BK channels are competent to contribute to resting K + efflux at normal cell membrane potentials with resting cytosolic Ca 2+ concentrations and likely play a critical physiological role in supporting normal secretory function in all secretory epithelial cells.L arge-conductance, voltage-and Ca 2+ -regulated BK-type channels are widely expressed proteins, found not only in excitable cells, such as neurons, muscle, and endocrine cells, but also nonexcitable cells, including salivary (1) and lacrimal gland (2) acinar cells, and colonic crypt cells (3). Given the almost ubiquitous expression of BK channels among cells that play quite distinct physiological roles, it is particularly important to define the specific properties of BK channels in a given cell type and determine what the specific physiological role played by BK channels in a given cell may be. A hallmark of BK channels is their dual regulation by both membrane voltage and cytosolic Ca 2+ (4), both properties embedded within the tetramer of pore-forming α-subunits of each BK channel (5). However, the specific range of voltages over which a BK channel is active at a given Ca 2+ concentration is markedly dependent on the identity of regulatory subunits that can coassemble with the α-subunit in the mature channel complex. Of the two families of known BK regulatory subunits, β (6-11) and γ (12-14), an important feature of many of these subunits is the ability to shift the range of activation voltages at a given Ca 2+ . Although there is growing information about the loci of expression and functional roles of BK channels containing specific β-subunits (15), much less is known about those BK channels containing the γ1 (LRRC26, leucine-rich-repeat-containing subunit 26) subunit. However, LRRC26 is particularly fascinating because it causes the largest shift in BK gating (approximately −120 mV) of any known non-pore-forming regulatory subunit, resulting in BK channels that...