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N-terminal inactivation domains of beta subunits are protected from trypsin digestion by binding within the antechamber of BK channels.

ABSTRACT: N termini of auxiliary beta subunits that produce inactivation of large-conductance Ca(2+)-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 beta2 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 beta subunit N termini. First, we show that specific mutations in the beta2 inactivation segment can speed up digestion by trypsin under closed-channel conditions, supporting the idea that the beta2 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 beta2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity. In contrast, the beta 3a N terminus is digested over 10-fold more quickly than the beta2 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.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC2654086 | biostudies-other | 2009 Mar

REPOSITORIES: biostudies-other

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Project description:Voltage/Ca²?(i)-gated, large conductance K+ (BK) channels result from tetrameric association of ? (slo1) subunits. In most tissues, BK protein complexes include regulatory ? subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK ? types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK ? modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK ?1 drastically increases the channel's apparent Ca²?(i) sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca2+ levels reached near the channel during depolarization-induced Ca2+ influx and myocyte contraction. The eventual BK channel activation generates outward K+ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca2+ influx. The BK ?1 regions responsible for the characteristic phenotype of ?1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant ?1, neuron-abundant ?4, or chimeras constructed by swapping ?1 and ?4 regions, and determined the contribution of specific ?1 regions to the BK phenotype. At Ca2+ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from ?1 and the remaining regions ("background") from ?4 showed a phenotype (V(half), ?(act), ?(deact)) identical to that of complexes containing wt ?1. This phenotype could not be evoked by complexes that included chimeras combining either ?1 TM1 or ?1 TM2 with a ?4 background. Likewise, ? "halves" (each including ?1 TM1 or ?1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between ?1 TMs via the EC loop is also necessary for proper channel function.

Project description:The KCNMB3 gene encodes one of a family of four auxiliary beta subunits found in the mammalian genome that associate with Slo1 alpha subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct beta3 subunits, beta3a-d. Three variants, beta3a-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 beta3 variants. Whereas beta1, beta2, and beta4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse beta3 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, beta3a and beta3b, of the four found in humans. Both human and mouse beta3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hbeta3b exhibits rapid inactivation, the putative mbeta3b does not inactivate. Furthermore, unlike hbeta3, the mbeta3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca(2+) 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 mbeta3b N terminus may not be a true orthologue of the hbeta3b N terminus and that both beta3c and beta3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous beta3 subunits may differ among mammalian species; second, the specific physiological roles of homologous beta3 subunits may differ among mammalian species; and, third, some beta3 variants may be primate-specific ion channel subunits.

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N-terminal inactivation domains of beta subunits are protected from trypsin digestion by binding within the antechamber of BK channels.

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