Regulation of large-conductance Ca2+- and voltage-gated K+ channels by electrostatic interactions with auxiliary β subunits

Large-conductance Ca2+- and voltage-gated K+ (BK KCa1.1) channel complexes include pore-forming Slo1 α subunits and often auxiliary β subunits, latter of which noticeably modify the channel’s pharmacological and gating characteristics. In the absence of intracellular Ca2+, β1 and β4 modestly shift the overall voltage dependence of the channel to the positive direction by decreasing the probability that the ion conduction gate is open without any allosteric influence from the channel’s voltage or Ca2+ sensors. This intrinsic open probability is also critically regulated by the intracellular-facing 329RKK331 segment of human Slo1 (hSlo1) downstream of the transmembrane segment S6 in association with two negatively charged residues in S6 (E321 and E324) (Tian et al., Proc Natl Acad Sci USA, 116, 8591-8596, 2019). This study examined how β1/β4 and the RKK segment function together to control the channel gate. With select mutations in the RKK segment, inclusions of β1 or β4 can dramatically increase the intrinsic gate opening probability and shift the overall voltage dependence of the channel to the negative direction by up to 200 mV without Ca2+. This remarkable shift is mediated at least in part by electrostatic interactions between the Slo1 RKK and β N-terminal segments as suggested by the results of double-mutant cycle analysis, ionic strength experiments, and molecular modelling. With or without auxiliary β subunits, the Slo1 RKK and E321/E324 segments are thus critical determinants of the intrinsic open probability of the ion conduction gate and changes in the electrostatic environment near the RKK-EE segments are a potential mechanism of pharmacological gating modifiers.

Regulation of Nav1.6 and Nav1.8 peripheral nerve Na+ channels by auxiliary β-subunits

Voltage-gated Na+ (Nav) channels are composed of a pore-forming α-subunit and one or more auxiliary β-subunits. The present study investigated the regulation by the β-subunit of two Na+ channels (Nav1.6 and Nav1.8) expressed in dorsal root ganglion (DRG) neurons. Single cell RT-PCR was used to show that Nav1.8, Nav1.6, and β1–β3 subunits were widely expressed in individually harvested small-diameter DRG neurons. Coexpression experiments were used to assess the regulation of Nav1.6 and Nav1.8 by β-subunits. The β1-subunit induced a 2.3-fold increase in Na+ current density and hyperpolarizing shifts in the activation (−4 mV) and steady-state inactivation (−4.7 mV) of heterologously expressed Nav1.8 channels. The β4-subunit caused more pronounced shifts in activation (−16.7 mV) and inactivation (−9.3 mV) but did not alter the current density of cells expressing Nav1.8 channels. The β3-subunit did not alter Nav1.8 gating but significantly reduced the current density by 31%. This contrasted with Nav1.6, where the β-subunits were relatively weak regulators of channel function. One notable exception was the β4-subunit, which induced a hyperpolarizing shift in activation (−7.6 mV) but no change in the inactivation or current density of Nav1.6. The β-subunits differentially regulated the expression and gating of Nav1.8 and Nav1.6. To further investigate the underlying regulatory mechanism, β-subunit chimeras containing portions of the strongly regulating β1-subunit and the weakly regulating β2-subunit were generated. Chimeras retaining the COOH-terminal domain of the β1-subunit produced hyperpolarizing shifts in gating and increased the current density of Nav1.8, similar to that observed for wild-type β1-subunits. The intracellular COOH-terminal domain of the β1-subunit appeared to play an essential role in the regulation of Nav1.8 expression and gating.

N-terminal Inactivation Domains of β Subunits Are Protected from Trypsin Digestion by Binding within the Antechamber of BK Channels

N termini of auxiliary β subunits that produce inactivation of large-conductance Ca2+-activated K+ (BK) channels reach their pore-blocking position by first passing through side portals into an antechamber separating the BK pore module and the large C-terminal cytosolic domain. Previous work indicated that the β2 subunit inactivation domain is protected from digestion by trypsin when bound in the inactivated conformation. Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber. Here, we provide additional tests of this model and examine its applicability to other β subunit N termini. First, we show that specific mutations in the β2 inactivation segment can speed up digestion by trypsin under closed-channel conditions, supporting the idea that the β2 N terminus is protected by binding within the antechamber. Second, we show that cytosolic channel blockers distinguish between protection mediated by inactivation and protection under closed-channel conditions, implicating two distinct sites of protection. Together, these results confirm the idea that β2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity. In contrast, the β3a N terminus is digested over 10-fold more quickly than the β2 N terminus. Analysis of factors that contribute to differences in digestion rates suggests that binding of an N terminus within the antechamber constrains the trypsin accessibility of digestible basic residues, even when such residues are positioned outside the antechamber. Our analysis indicates that up to two N termini may simultaneously be protected from digestion. These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation.