Slo3 K+ channels: voltage and pH dependence of macroscopic currents

The mouse Slo3 gene (KCNMA3) encodes a K(+) channel that is regulated by changes in cytosolic pH. Like Slo1 subunits responsible for the Ca(2+) and voltage-activated BK-type channel, the Slo3 alpha subunit contains a pore module with homology to voltage-gated K(+) channels and also an extensive cytosolic C terminus thought to be responsible for ligand dependence. For the Slo3 K(+) channel, increases in cytosolic pH promote channel activation, but very little is known about many fundamental properties of Slo3 currents. Here we define the dependence of macroscopic conductance on voltage and pH and, in particular, examine Slo3 conductance activated at negative potentials. Using this information, the ability of a Horrigan-Aldrich-type of general allosteric model to account for Slo3 gating is examined. Finally, the pH and voltage dependence of Slo3 activation and deactivation kinetics is reported. The results indicate that Slo3 differs from Slo1 in several important ways. The limiting conductance activated at the most positive potentials exhibits a pH-dependent maximum, suggesting differences in the limiting open probability at different pH. Furthermore, over a 600 mV range of voltages (-300 to +300 mV), Slo3 conductance shifts only about two to three orders of magnitude, and the limiting conductance at negative potentials is relatively voltage independent compared to Slo1. Within the context of the Horrigan-Aldrich model, these results indicate that the intrinsic voltage dependence (z(L)) of the Slo3 closed-open equilibrium and the coupling (D) between voltage sensor movement are less than in Slo1. The kinetic behavior of Slo3 currents also differs markedly from Slo1. Both activation and deactivation are best described by two exponential components, both of which are only weakly voltage dependent. Qualitatively, the properties of the two kinetic components in the activation time course suggest that increases in pH increase the fraction of more rapidly opening channels.     

Interactions between β Subunits of the KCNMB Family and Slo3: β4 Selectively Modulates Slo3 Expression and Function

Background

The pH and voltage-regulated Slo3 K⁺ channel, a homologue of the Ca²⁺- and voltage-regulated Slo1 K⁺ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary β subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct β subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels.

Methodology/Principal Findings

To examine the ability of the KCNMB family of auxiliary β subunits to regulate Slo3 function, we co-expressed Slo3 and each β subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The β4 subunit produced an 8–10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither β1, β2, nor β3 mimicked the ability of β4 to increase surface expression, although biochemical tests suggested that all four β subunits are competent to coassemble with Slo3. Fluorescence microscopy from β4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that β4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that β4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm.

Conclusions/Significance

These results argue that, for native mouse Slo3 channels, the β4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 α subunit.     

Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels

An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.     

Inactivating BK channels in rat chromaffin cells may arise from heteromultimeric assembly of distinct inactivation-competent and noninactivating subunits.

Inactivating and noninactivating variants of large-conductance, Ca2+-dependent, voltage-dependent BK-type channels are found in rat chromaffin cells and are largely segregated into different cells. Here we test the hypothesis that, within the population of cells that express inactivating BK current (BKi current), the BKi channels are largely heteromultimers composed of inactivation-competent subunits (bk(i)) and noninactivating subunits (bk(s)). Several independent types of evidence support this view. The gradual removal of inactivation by trypsin is consistent with the idea that in most cells and patches there are, on average, about two to three inactivation domains per channel. In addition, several aspects of blockade of BKi current by charybdotoxin (CTX) are consistent with the idea that BKi channels contain differing numbers (one to four) of relatively CTX-resistant bk(i) subunits. Finally, the frequency of occurrence of noninactivating BKs channels in patches with predominantly inactivating BKi channels is consistent with the binomial expectations of random, independent assembly of two distinct subunits, if most cells have, on average, about two to three bk(i) subunits per channel. These results suggest that the phenotypic properties of BKi currents and the resulting cellular electrical excitability may exhibit a continuum of behavior that arises simply from the differential expression of two distinct subunits.     

Short term cyclin D1 overexpression induces centrosome amplification, mitotic spindle abnormalities, and aneuploidy

In normal cells, cyclin D1 is induced by growth factors and promotes progression through the G(1) phase of the cell cycle. Cyclin D1 is also an oncogene that is thought to act primarily by bypassing the requirement for mitogens during the G(1) phase. Studies of clinical tumors have found that cyclin D1 overexpression is associated with chromosome abnormalities, although a causal effect has not been established in experimental systems. In this study, we found that transient expression of cyclin D1 in normal hepatocytes in vivo triggered dysplastic mitoses, accumulation of supernumerary centrosomes, abnormalities of the mitotic spindle, and marked chromosome changes within several days. This was associated with up-regulation of checkpoint genes p53 and p21 as well as hepatocyte apoptosis in the liver. Transient transfection of cyclin D1 also induced centrosome and mitotic spindle abnormalities in breast epithelial cells, suggesting that this may be a generalized effect. These results indicate that cyclin D1 can induce deregulation of the mitotic apparatus and aneuploidy, effects that could contribute to the role of this oncogene in malignancy.     

<Emphasis Type="Italic">ANKH</Emphasis>variants associated with ankylosing spondylitis: gender differences

The ank (progressive ankylosis) mutant mouse, which has a nonsense mutation in exon 12 of the inorganic pyrophosphate regulator gene (ank), exhibits aberrant joint ankylosis similar to human ankylosing spondylitis (AS). We previously performed family-based association analyses of 124 Caucasian AS families and showed that novel genetic markers in the 5' flanking region of ANKH (the human homolog of the murine ank gene) are modestly associated with AS. The objective of the present study was to conduct a more extensive evaluation of ANKH variants that are significantly associated with AS and to determine whether the association is gender specific. We genotyped 201 multiplex AS families with nine ANKH intragenetic and two flanking microsatellite markers, and performed family-based association analyses. We showed that ANKH variants located in two different regions of the ANKH gene were associated with AS. Results of haplotype analyses indicated that, after Bonferroni correction, the haplotype combination of rs26307 [C] and rs27356 [C] is significantly associated with AS in men (recessive/dominant model; P = 0.004), and the haplotype combination of rs28006 [C] and rs25957 [C] is significantly associated with AS in women (recessive/dominant model; P = 0.004). A test of interaction identified rs26307 (i.e. the region that was associated in men with AS) as showing a difference in the strength of the association by gender. The region associated with AS in women only showed significance in the test of interaction among the subset of families with affected individuals of both genders. These findings support the concept that ANKH plays a role in genetic susceptibility to AS and reveals a gender–genotype specificity in this interaction.