Properties of Slo1 K+ channels with and without the gating ring

High-conductance Ca²⁺- and voltage-activated K⁺ (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca²⁺ and Mg²⁺ sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca²⁺- and Mg²⁺-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca²⁺ and Mg²⁺ activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.Slo1 channels are expressed in most human tissues and play key roles in many important physiological processes, including smooth muscle contraction, neurotransmitter release, neuronal excitability, hair cell tuning, and action potential termination (1–6). Slo1 channels also are named BK (Big K⁺) or MaxiK channels because of their high single-channel conductance (∼300 pS in 150-mM symmetrical K⁺). Slo1 channels are activated synergistically by both depolarization and intracellular calcium (7–9), linking these two activators in a negative feed-back system to restore negative membrane potential which, in turn, closes voltage-activated Ca²⁺ channels. The dual regulation by voltage and calcium led Hille (10) to predict that BK channels function like the classical Hodgkin–Huxley delayed rectifier channel, except that the range of voltage activation was set by the intracellular Ca²⁺ concentration. The cloning (11) and analysis of the Slo1 channel structure seemed to validate this prediction, in that Slo1 appeared to be modular in its construction, having a core domain containing a voltage sensor controlling a K⁺-selective pore and a long C-terminal tail forming a gating ring structure comprised of four pairs of regulators of the conductance of K⁺ (RCK) domains for sensing and transducing the effect of Ca²⁺ binding to the core.One of the four identical α subunits that assemble to form the Slo1 WT channel (Slo1-WT) is shown in Fig. 1Top. For the mbr5 cDNA (12) used in this study, the “core” consists of 342 residues including seven transmembrane segments (S0–S6) and the S6–RCK1 linker sequence, which is attached to a long tail of 827 residues. The tail sequence of Slo1-WT is distinct from the cytoplasmic domains of other members of the K⁺ channel extended family. Structure–function studies of the tail have shown the existence of two high-affinity Ca²⁺ binding sites (13, 14) and one low-affinity Mg²⁺ site (14, 15). Modulation of the channel also occurs by additional biological factors, including protons, heme, carbon monoxide, phosphorylation, and oxidation (16–20), all of which may function via their interaction with the tail. Thus, the large tail accommodates a variety of regulatory domains which sense different intracellular factors, leading to pushing or tugging against the core to facilitate or inhibit channel gating. These complicated allosteric interactions between core and tail almost certainly involve several transduction pathways (21–23), all of which alter the properties of the core. Thus, a logical starting point to begin investigating the allosteric interactions would be to understand the baseline properties of the isolated core. However, this approach has been hampered by the inability to express functional cores in the absence of the tail. Previous analysis of truncated expression constructs of Slo1 channels found that their processing stalls in the endoplasmic reticulum (ER), they are not assembled into tetramers, they fail to be exported to the plasma membrane, or they are nonfunctional (24). We now show that core constructs without gating rings can be expressed by leaving a short region required for subunit tetramerization and by appending a small tail domain which facilitates processing and efficient export to the plasma membrane. Thus, we now are able to investigate gating in the absence of a gating ring.Open in a separate windowFig. 1.Slo1 channel constructs used in this study. The Slo1 channel constructs used in this study are based on the mouse mbr5 cDNA (12) and the mouse Shaker family Kv1.4 channel (25). The “Slo1 core and tail” refers to the first 342 and the last 827 amino acid residues. The “Kv1.4 tail” refers to the last 74 amino acid residues of Kv1.4. The different channel constructs are designated as follows: Slo1-WT is Slo1 full-length WT; Slo1C-KvT is a Slo1 core with a 74-residue Kv1.4 tail; Slo1C-Kv-minT is a Slo1 core with a Kv1.4 11-residue mini tail; Slo1C-KvT_NAFQ is a Slo1 core with a 74-residue Kv1.4 tail with NAFQ substituted for KKFR in the tail; Slo1C-KvT R207E is Slo1C-KvT with R207E in S4 in the core.