Species-specific Differences among KCNMB3 BK β3 Auxiliary Subunits: Some β3 N-terminal Variants May Be Primate-specific Subunits

The KCNMB3 gene encodes one of a family of four auxiliary β subunits found in the mammalian genome that associate with Slo1 α subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct β3 subunits, β3a–d. Three variants, β3a–c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse β3 variants. Whereas β1, β2, and β4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse β3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, β3a and β3b, of the four found in humans. Both human and mouse β3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hβ3b exhibits rapid inactivation, the putative mβ3b does not inactivate. Furthermore, unlike hβ3, the mβ3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca2+ concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mβ3b N terminus may not be a true orthologue of the hβ3b N terminus and that both β3c and β3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous β3 subunits may differ among mammalian species; second, the specific physiological roles of homologous β3 subunits may differ among mammalian species; and, third, some β3 variants may be primate-specific ion channel subunits

Inactivation of BK Channels by the NH2 Terminus of the β2 Auxiliary Subunit: An Essential Role of a Terminal Peptide Segment of Three Hydrophobic Residues

An auxiliary β2 subunit, when coexpressed with Slo α subunits, produces inactivation of the resulting large-conductance, Ca2+ and voltage-dependent K+ (BK-type) channels. Inactivation is mediated by the cytosolic NH2 terminus of the β2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH2 terminus in the inactivation process. The β2 NH2 terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2–4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH2-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process

Two classes of regulatory subunits coassemble in the same BK channel and independently regulate gating

High resolution proteomics increasingly reveals that most native ion channels are assembled in macromolecular complexes. However, whether different partners have additive or cooperative functional effects, or whether some combinations of proteins may preclude assembly of others are largely unexplored topics. The large conductance Ca(2+)-and-voltage activated potassium channel (BK) is well-suited to discern nuanced differences in regulation arising from combinations of subunits. Here we examine whether assembly of two different classes of regulatory proteins, β and γ, in BK channels is exclusive or independent. Our results show that both γ1 and up to four β2-subunits can coexist in the same functional BK complex, with the gating shift caused by β2-subunits largely additive with that produced by the γ1-subunit(s). The multiplicity of β:γ combinations that can participate in a BK complex therefore allow a range of BK channels with distinct functional properties tuned by the specific stoichiometry of the contributing subunits

Effects of polymer additives in the bulk of turbulent thermal convection

We present experimental evidence that a minute amount of polymer additives can significantly enhance heat transport in the bulk region of turbulent thermal convection. The effects of polymer additives are found to be the \textit{suppression} of turbulent background fluctuations that give rise to incoherent heat fluxes that make no net contribution to heat transport, and at the same time to \textit{increase} the coherency of temperature and velocity fields. The suppression of small-scale turbulent fluctuations leads to more coherent thermal plumes that result in the heat transport enhancement. The fact that polymer additives can increase the coherency of thermal plumes is supported by the measurements of a number of local quantities, such as the extracted plume amplitude and width, the velocity autocorrelation functions and the velocity-temperature cross-correlation coefficient. The results from local measurements also suggest the existence of a threshold value for the polymer concentration, only above which can significant modification of the plume coherent properties and enhancement of the local heat flux be observed. Estimation of the plume emission rate suggests that the second effect of polymer additives is to stabilize the thermal boundary layers.Comment: 8 figures, 11 page

Direct Observation of a Preinactivated, Open State in BK Channels with β2 Subunits

Proteins arising from the Slo family assemble into homotetramers to form functional large-conductance, Ca2+- and voltage-activated K+ channels, or BK channels. These channels are also found in association with accessory β subunits, which modulate several aspects of channel gating and expression. Coexpression with either of two such subunits, β2 or β3b, confers time-dependent inactivation onto BK currents. mSlo1+β3b channels display inactivation that is very rapid but incomplete. Previous studies involving macroscopic recordings from these channels have argued for the existence of a second, short-lived conducting state in rapid equilibrium with the nonconducting, inactivated conformation. This state has been termed “pre-inactivated,” or O*. β2-mediated inactivation, in contrast, occurs more slowly but is virtually complete at steady state. Here we demonstrate, using both macroscopic and single channel current recordings, that a preinactivated state is also a property of mSlo1+β2 channels. Detection of this state is enhanced by a mutation (W4E) within the initial β2 NH2-terminal segment critical for inactivation. This mutation increases the rate of recovery to the preinactivated open state, yielding macroscopic inactivation properties qualitatively more similar to those of β3b. Furthermore, short-lived openings corresponding to entry into the preinactivated state can be observed directly with single-channel recording. By examining the initial openings after depolarization of a channel containing β2-W4E, we show that channels can arrive directly at the preinactivated state without passing through the usual long-lived open conformation. This final result suggests that channel opening and inactivation are at least partly separable in this channel. Mechanistically, the preinactivated and inactivated conformations may correspond to binding of the β subunit NH2 terminus in the vicinity of the cytoplasmic pore mouth, followed by definitive movement of the NH2 terminus into a position of occlusion within the ion-conducting pathway

The Schur concavity, Schur multiplicative and harmonic convexities of the second dual form of the Hamy symmetric function with applications

AbstractFor x=(x1,x2,…,xn)∈R+n, the second dual form of the Hamy symmetric function is defined by Hn∗∗(x,r)=Hn∗∗(x1,x2,…,xn;r)=∏1≤i1<i2<⋯<ir≤n(∑j=1rxij)1r, where r∈{1,2,…,n} and i1,i2,…,in are positive integers.In this paper, we prove that Hn∗∗(x,r) is Schur concave, and Schur multiplicatively and harmonic convex in R+n. Some applications in inequalities and reliability theory are presented

Divalent Cation Sensitivity of BK Channel Activation Supports the Existence of Three Distinct Binding Sites

Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming α subunit. Two mechanisms account for physiological regulation of BK channels by μM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10–300 μM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels