July 21, 2009;
Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.
The pH and voltage-regulated Slo3 K(+) channel, a homologue of the Ca(2+)- 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 beta subunits regulating Slo1
channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1
Channels. To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 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 beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT
-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes
. These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1
[+] show captions
Figure 1. KCNMB4 increases Slo3 macroscopic conductance in Xenopus oocytes over 8-fold.Recordings were done in inside-out patches 5 days after cRNA injection. In A–D, an identical amount of mSlo3 cRNA was injected into oocytes all from the same batch. In A, mSlo3 currents were activated with the indicated voltage-protocol either at pH 7.6 (left) or pH 8.5 (right). In B, currents resulting from coexpression of mSlo3 and hβ4 were recorded as in A. In C, mean steady-state and tail conductances measured as a function of voltage at pH 8.5 were determined for both Slo3 (n = 9) and Slo3+hβ4 (n = 8). Steady state current for Slo3+hβ4 at voltages over +100 mV typically exceeded the range of the recording system. In D, GV curves were normalized to the maximum conductance estimated from a fit of a Boltzmann function (Eq. 1) to each curve. Numbers of patches for Slo3 steady-state current and Slo3+hβ4 tail current are the same as in C. Slo3 tail GV curves were obtained from 5 patches with current amplitudes sufficient to allow meaningful Boltzmann fitting, while Slo3+hβ4 steady state GV curves were from 4 patches with current amplitudes at +200 mV within the range of the recording system. In E and F, all patches were from a separate batch of oocytes injected with identical amounts of Slo3 cRNA and maintained for 5 days at 17°C. In E, steady-state current amplitudes measured at pH 8.5 and +150 mV are compared for Slo3 alone and for coexpression with other BK β subunits. In F, Slo3 +β2 current measured at pH 8.5 and +150 mV was robustly increased after trypsin application. A pre-pulse at −160 mV for 100 ms was used to drive channels to resting states before activation.
Figure 2. Coexpression of Slo3 with β4 does not appreciably increase single channel conductance or effective Po.In A, traces show openings from a patch with a single Slo3+β4 channel at +150 mV with pH 8.5 (filtering at 10 kHz). In B, a total amplitude histogram of all digitized current values from a 995 ms record of Slo3+β4 single channel activity is plotted, showing a substantial number of current values between the closed level (0 pA) and the largest open level (∼18 pA). Histograms were fit with a 3-component Gaussian function to estimate the fraction and amplitude of the closed level, the smaller current level (gS) and the larger current level (gL). In C, the gS fraction of all open current levels is plotted as a function of voltage (3–5 patches). In D, the mean conductance for gS, gL, and the overall mean is plotted as a function of voltage. In E, σ2 is plotted as a function of mean current level for current recorded from a single patch at 5 different voltages. For each voltage, a set of 100 repeated steps to the nominal voltage was used to activate currents from which σ2 and mean current, I, were determined for each time point in the average. The σ2/I relationships at multiple voltages were fit simultaneously by σ2(I,V) = g*V*I - I2/N with best fit values indicated by the solid lines, yielding g = 82.7±1.5 pS and N = 1284.5±73.6 channels. In F, the mean conductance estimated from single channel measurements at +150 mV (n = 5) and the single channel conductance estimated from σ2/I estimates (n = 6) are compared, along with previous estimates for Slo3 alone (dotted line). In G, estimates of Po for Slo3+β4 patches at pH 8.5 obtained either from σ2/I estimates (black circles, n = 3–6) or from the mean single channel current estimate (red diamond, n = 5) are plotted as a function of voltage and compared to previous estimates from Slo3 (dotted line).
Figure 3. β4 increases surface expression of Slo3.In A, oocytes from the same batch were injected either with mSlo3::eYFP (eYFP inserted between RCK domains) alone, mSlo3::eYFP+hβ4, or with H2O. Confocal sections revealed weak fluoresence in oocytes injected only with mSlo3 alone, while hβ4 strongly increased surface fluoresence. For illustrative purposes, an oocyte of each category was positioned together and viewed either with fluorescent (left) or normal illumination (right). In B, oocytes were injected with Slo3-eYFP (eYFP attached to C-terminus) with or without hβ4. As in A, hβ4 substantially increased surface fluorescence. In C and D, Slo3 was FLAG tagged. In C, Slo3 either in samples of total protein or surface protein expressed either alone or together with β4 in Xenopus oocytes was detected with Western blotting. Total Slo3 protein was purified by immunoprecipitation with anti-FLAG M2 agarose. After “blotting” of the immunoprecipitated proteins, 1∶200 anti-Slo3 antibody was used as the primary antibody. Integrated density of the immunoblots was quantified using ImageJ 1.40 g software (http://rsb/info.nih/gov/ij/). For total protein samples, the amount of Slo3 protein in the presence of β4 is 1.2-fold of that in the absence of β4. The corresponding ratio for surface protein is 12. In D, total and surface expression of β4+/−Slo3 are compared. Monoclonal anti-mβ4 antibody (1∶200) served as the primary antibody. The ratio of β4 amount in the presence of Slo3 compared to that without Slo3 is 1 or 11, for total protein or surface protein samples, respectively.
Figure 4. β4 may specifically co-exist with Slo3 in mouse sperm.In A, membrane proteins from WT mouse cauda epididymal sperm were detected with anti-mSlo3 antibody as primary antibody. In B, a section from wild-type mouse testis showing well-defined seminiferous tubules is shown with normal (B1) and fluorescent (B2) illumination. Background fluorescence is limited to spaces between seminiferous tubules. A section of testis from a β4 KO mouse is shown under normal (B3) and fluorescent (B4) illumination revealing prevalent GFP fluorescence within seminiferous tubules. In C, similar comparisons are shown for sperm collected from caudal epididymis of both WT (C1, C2) and β4 KO mice (C3, C4). No significant fluorescence was detected from blood cells of β4 KO mice (C5, C6). In D, RNA - threshold cycle relationship reveals that the abundance of β4 and Slo3 mRNA in testes is similar. Each data point is an average from three experiments. The solid lines are linear fits with the slope values as follows: β-actin, −3.26; Slo1, −3.62; Slo3, −3.51; β1, −2.94; β2, −2.62; β3, −3.34; β4, −3.27. In E (testes, n = 3) and F (sperm, n = 3), the relative abundance of each tested subunit compared to Slo3 is displayed. Abundance was calculated from 2−dCt, with dCt = Ct(subunit) - Ct(Slo3). Ct(subunit) stands for the threshold cycle number of the corresponding subunit, and Ct(Slo3) is the average threshold cycle number for Slo3 obtained from three parallel experiments for either testes or sperm. Scale bars: 50 µm in B, 10 µm in C.
Figure 5. β4 regulates Slo3 even in the presence of Slo1.In A–D, currents were obtained from oocytes injected with Slo3 and Slo1 at 1∶1 ratio, together with hβ4 (Slo3/Slo1 = 1+hβ4). Example traces in A–C were recorded from the same patch in the order from A to C. Tail currents were recorded at −120 mV for all cases. In A, the trace shows exclusively Ca2+-dependent current. In B, currents were recorded with 0 Ca2+ at +180 mV either at pH 7.0 or pH 8.5 to reveal the pH-dependent current. In C, current was recorded at +180 mV under 0 Ca2+ at pH 8.5 with 200 nM paxilline applied to block Slo1 channels. The dotted line indicates the pH-dependent current revealed by subtraction of current recorded at pH 7.0 from that recorded at pH 8.5 in B. In D, the amplitude of the paxilline-resistant current is compared to that of the pH-dependent current. Both paxilline-resistant conductance and pH-activated conductance were normalized to that of the Ca2+-dependent current from the same patch. In E, the activation time constants at +180 mV of the pH-dependent currents obtained from different Slo3/Slo1/β4 combinations are compared. In F, the activation time constants at +60 mV of the Ca2+-dependent currents obtained from different Slo3/Slo1/β4 combinations are compared. In G, the pH-dependent conductance relative to the Ca2+-dependent conductance in each combination is shown.