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The Journal of Neuroscience, July 1, 2000, 20(13):4890-4903

Rectification and Rapid Activation at Low Ca²⁺ of Ca²⁺-Activated, Voltage-Dependent BK Currents: Consequences of Rapid Inactivation by a Novel  Subunit

Xiao-Ming Xia, Jiu-Ping Ding, Xu-Hui Zeng, Kai-Lai Duan, and Christopher J. Lingle

Washington University School of Medicine, Departments of Anesthesiology, and Anatomy and Neurobiology, St. Louis, Missouri 63110

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A family of accessory  subunits significantly contributes to the functional diversity of large-conductance, Ca²⁺- and voltage-dependent potassium (BK) channels in native cells. Here we describe the functional properties of one variant of the  subunit family, which confers properties on BK channels totally unlike any that have as yet been observed. Coexpression of this subunit (termed 3) with Slo  subunits results in rectifying outward currents and, at more positive potentials, rapidly inactivating (~1 msec) currents. The underlying rapid inactivation process results in an increase in the apparent activation rate of macroscopic currents, which is coupled with a shift in the activation range of the currents at low Ca²⁺. As a consequence, the currents exhibit more rapid activation at low Ca²⁺ relative to any other BK channel subunit combinations that have been examined. In part because of the rapid inactivation process, single channel openings are exceedingly brief. Although variance analysis suggests a conductance in excess of 160 pS, fully resolved single channel openings are not observed. The inactivation process results from a cytosolic N-terminal domain of the 3 subunit, whereas an extended C-terminal domain does not participate in the inactivation process. Thus, the 3 subunit appears to use a rapid inactivation mechanism to produce a current with a relatively rapid apparent activation time course at low Ca²⁺. The 3 subunit is a compelling example of how the  subunit family can finely tune the gating properties of Ca²⁺- and voltage-dependent BK channels.

Key words: accessory subunits; K⁺ channels; BK channels; Ca²⁺- and voltage-gated K⁺ channels; mSlo channels; inactivation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ca²⁺- and voltage-dependent K⁺ (BK-type) channels, like the voltage-gated K⁺ channels, are composed of four homologous  subunit peptides that contribute to the ion channel pore (Atkinson et al., 1991; Adelman et al., 1992; Butler et al., 1993; Shen et al., 1994), along with  accessory subunits that regulate many important aspects of BK channel function (McManus et al., 1995; Wallner et al., 1995, 1999; Dworetzky et al., 1996; Tseng-Crank et al., 1996; Nimigean and Magleby, 1999; Xia et al., 1999). Typically, BK channels exhibit a stereotypic, large unitary conductance but exhibit considerable functional diversity in regards to kinetic behavior, apparent Ca²⁺ dependence, and pharmacology (McManus, 1991). Thus, BK channels can play a variety of physiological roles well suited to the demands of the cells in which they are found. The functional diversity can, in part, be accounted for by various splice variants of the  subunit encoded by the Slo loci (Adelman et al., 1992; Tseng-Crank et al., 1994; Jones et al., 1998; Ramanathan et al., 1999). However, the family of accessory  subunits may, in fact, play a more important role in defining the phenotypic properties of BK channels, including the effective gating range and inactivation behavior (McManus et al., 1995; Jones et al., 1998; Wallner et al., 1999; Xia et al., 1999), because the functional variation arising from Slo splice variants appears rather modest (Adelman et al., 1992; Tseng-Crank et al., 1994; Saito et al., 1997; Jones et al., 1998; Ramanathan et al., 1999). To date, including the subunit studied in this paper, four distinct members of a mammalian  subunit family have been identified [KCNMB1 (1) (Knaus et al., 1994); KCNMB2 (2) (Wallner et al., 1999; Xia et al., 1999); KCNMB3 (3) (Riazi et al., 1999; Brenner et al., 2000; Uebele et al., 2000); KCNMB4 (4) (Wickenden et al., 1999; Brenner et al., 2000; Meera et al., 2000; Wallner et al., 2000)], each of which appears to confer unique functional properties on the resulting BK channels. Definition of the functional properties of the  subunits is an essential step in understanding the diversity of phenotypic properties of BK channels in native cells.

Here, we describe the functional properties of new  subunit (termed 3) that confers onto BK channels properties totally unlike any that have as yet been described. Genomic sequence encoding a portion of this subunit has been described recently (Riazi et al., 1999), although the subunit we have identified contains differences in the N terminus from that proposed. Coexpression of this new subunit with Slo  subunits results in an extremely rapidly inactivating, Ca²⁺- and voltage-dependent current with pharmacological properties characteristic of previously described BK channels. However, the currents have several properties unlike other BK currents, including rectification of outward current and extremely flickery, unresolvable single channel openings. The effects of this particular  subunit family member illustrate an extreme example of the diverse ways ion channel function can be altered by an accessory subunit.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of the full-length cDNA clone of 3. Using homology searching of human expressed sequenced tagged (EST) databases based on BK 1 (Knaus et al., 1994), an avian  subunit (Oberst et al., 1997), and 2 (Wallner et al., 1999; Xia et al., 1999) sequences, several partial cDNA sequences (GenBank accession numbers AA761761, AA195511, AA823768, and AA236968) suggestive of a new  subunit family member were identified. These cDNA clones were obtained from Genome Systems (St. Louis, MO) and resequenced with BigDye Terminator Kit (Applied Biosystems, Foster City, CA). The four EST cDNA clones exhibited sufficient overlap to define most of the 3 sequence, including the 3' terminus, but terminated at a position aligning within TM1 defined by the other  subunits. Thus, the EST cDNA clones failed to identify a full-length clone and lacked the start codon at the 5' end. To search for the 5' coding sequence, a nested PCR was performed on human heart cDNA library (Stratagene, La Jolla, CA), from which several of the EST clones had been identified. The two rounds of PCR were performed with a vector-specific primer and a 3-specific primer (first PCR, Lacmer and 5'-CATCATGGCAAACCCCAGCATCAC-3'; second PCR, T3 primer and 5'-GAAGATCTCCCAGCATCACGGCTCGGTCCTCTCC-3', with pfu polymerase, 30-36 cycles, 96°C for 30 sec, 50°C for 45 sec, and 72°C for 2 min). The PCR products were then purified and digested with EcoRI and BglII overnight and then gel purified and subcloned in pSK plasmid (Stratagene). Sequencing revealed that one pSK clone contained an extra 500 base pairs upstream of the 3 sequence. The continuity of this sequence to the rest of 3 was justified by the fact that 46 base pairs at the 3' end of the PCR-generated fragment were identical to sequence obtained from the partial 3 sequence identified by the EST sequences. This new sequence identified by PCR define 19 additional amino acids at the 3 N terminus that precede an upstream stop codon in the same reading frame.

Tissue distribution of the 3 message. Northern blots were performed on membranes purchased from Clontech (Palo Alto, CA) containing ~2 µg of poly(A⁺) RNA per lane from various human tissues. The PCR fragment of full-length 3 was ³²P-labeled as the hybridization probe. The vendor adjusted the RNA loading of each lane based on previous blots using human -actin cDNA as probe. The experimental conditions used were described previously (Xia et al., 1999).

Expression constructs. The Xenopus oocyte expression vector pBF was used to subclone all of the DNA constructs (Xia et al., 1998). The mSlo  subunit [GenBank accession number L16912 (Butler et al., 1993)] was digested with ClaI and filled in to generate blunt ends at the 5' end of mSlo; a 3' SalI site was used to obtain a blunt-end SalI fragment. The resultant mSlo was then subcloned into a HpaI-SalI-digested pBF vector. The full-length 3 expression clone was generated by overlapping PCR and DNA manipulation. The 3' part was obtained by subcloning 3 EcoRI fragment from EST clone AA761761 to the EcoRI-digested vector portion of AA195511, which contains partial 3 3' end sequence. This intermediate construct, which is a partial 3 sequence lacking the 5' end, was screened by sequencing to define the correct orientation. The complete full-length 3 clone was then acquired by overlapping PCR (Xia et al., 1999) (first round PCR primer, reaction A, 5'-AGGGATCCACTGCCAATGACAGCCTTTCCTG-3' and 5'-GGCAGCCTCTTGTGCACATCTAGTGGGTCTCCATC-3'; reaction B, 5'-GATGGAGACCCACTAGATGTGCACAAGAGGCTGAA-3' and 5'-TACTAGTCGACTTAAGATTTCTCTGCTCTTCCTT-3'; second round PCR primer, 5'-AGGGATCCACTGCCAATGACAGCCTTTCCTG-3' and 5'-TACTAGTCGACTTAAGATTTCTCTGCTCTTCCTT-3'; pfu polymerase, 30-36 cycles, 96°C for 30 sec, 52°C for 45 sec, and 72°C for 2 min). The final overlapping PCR product was digested with BamHI and SalI and subcloned in the BamHI-SalI pBF oocyte expression vector (Xia et al., 1998). Both strands of DNA were sequenced for verification.

3 N-terminal or C-terminal deletion constructs D3, D4, and D5 were generated by pfu PCR with specific 3 primers (D3 primer, D4 primer, and D5 primer). The 2 N terminus 3 chimera (construct D1) and 3 N terminus 2 chimera (construct D20) were generated by overlapping PCR (Xia et al., 1999). To study the other potential 5' alternative h3 variant (Riazi et al., 1999), we amplified the 5' end from HEK293 genomic DNA using pfu PCR and then used overlapping PCR to link the 5' sequence with the rest of the 3' h3 sequence. In the first round PCR, two PCR reactions were performed using either human genomic DNA (A) or h3 DNA (B) as templates (reaction A primers, 5'-ATATATCTAGACA CAGGTAGGCAGCAAATGAGATTATCC-3' and 5'-GGCAGCCTAT TGTGCACATCTAGTGGGTCTCCATC-3'; reaction B primers, 5'-GATGGAGACCCACTAGATGTGCACAAGAGGCTGCC-3' and 5'-CTACGTCGACTTAAGATTTCTCTGCTCTTCCTT-3'; pfu DNA polymerase, 96°C for 30 sec, 50°C for 30 sec, and 72°C for 1 min, 36 cycles). The second round PCR was performed using both reaction A and reaction B as templates (primer, 5'-ATATATCTAGACACAGGTAGGCAGCAAATGAGATTATCC-3' and 5'-CTACGTCGACTTAAGATTTCTCTGCTCTTCCTT-3'; pfu DNA polymerase, 96°C for 30 sec, 50°C for 30 sec, and 72°C for 2 min, 36 cycles). The reaction product was then purified, digested with XbaI and SalI overnight, gel-purified, and subcloned into the XbaI-SalI pBF vector. Both strands of DNA were sequenced for verification. This molecule is referred to as Gmh3.

Expression in Xenopus oocytes. Methods of expression in Xenopus oocytes were as described previously (Xia et al., 1999). SP6 RNA polymerase was used to synthesize cRNA for oocyte injection after DNA was linearized with MluI (Xia et al., 1998). Fifty nanoliters of cRNA (10-20 ng/µl) was injected into stage IV Xenopus oocytes harvested 1 d before. To ensure a molar excess of  subunits to Slo  subunits, we regularly injected : at 1:1 or 1:2 ratios by weight.

During electrophysiological recordings, oocytes were maintained in ND96 [(96 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, and 5.0 mM HEPES, pH 7.5) supplemented with sodium pyruvate (2.5 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin 50 µg/ml]. Oocytes were used 1-7 d after injection of cRNA.

Electrophysiology and analysis. Currents were recorded in either inside-out or outside-out patches (Hamill et al., 1981). Patches used for determination of conductance-voltage (G-V) relationships contained large numbers of channels and were typically from oocytes maintained for 3-7 d. Generation of ensemble averages of channel openings was done with patches with fewer channels from oocytes maintained for 1-3 d before recording. Currents were typically digitized at 20-100 kHz. Macroscopic records were filtered at 5-20 kHz (Bessel low-pass filter; 3 dB) during digitization. Single channel records were filtered at 10 kHz (Bessel low-pass filter; 3 dB).

During seal formation, oocytes were bathed in ND96. After excision, patches were quickly moved into a flowing 0 Ca²⁺ solution. For inside-out recordings, the pipette extracellular solution was 140 mM potassium methanesulfonate, 20 mM KOH, 10 mM HEPES, and 2 mM MgCl₂, pH 7.0. Test solutions bathing the cytoplasmic face of the patch membrane contained 140 mM potassium methanesulfonate, 20 mM KOH, 10 mM HEPES, pH 7.0, and one of the following: 5 mM EGTA (for nominally 0 Ca²⁺, 0.5 µM, and 1 µM Ca²⁺ solutions), 5 mM HEDTA (for 4 and 10 µM Ca²⁺ solutions), or no added Ca²⁺ buffer (for 60 µM, 100 µM, 300 µM, and 5 mM Ca²⁺ solutions). The methanesulfonate solutions were calibrated to be identical to a set of chloride-containing solutions with free Ca²⁺ determined from a computer program (EGTAETC; E. McCleskey, Vollum Institute, Portland, OR). Calibration was also performed against a commercial set of Ca²⁺ standards [World Precision Instruments (WPI), Sarasota, FL], which yielded values essentially identical to our Cl-based standards. In all cases, the desired free Ca²⁺ was obtained by titrating the solution with calcium methanesulfonate until the electrode measurement of the methanesulfonate-based solution matched that of the chloride-based solution and the WPI calibration solution. Local perfusion of membrane patches was as described previously (Solaro and Lingle, 1992; Solaro et al., 1997).

Voltage commands and acquisition of currents was accomplished with pClamp 7.0 or 8.0 for Windows (Axon Instruments, Foster City, CA). Current values were measured using ClampFit (Axon Instruments), converted to conductances, and then fit with a nonlinear least squares fitting program. As described in Results, conductances were determined in three ways: from tail currents, from the peak current at a given activation potential, and from steady-state current at a given activation potential. G-V curves for activation were fit with a Boltzmann equation with the form:

(1)

where V_0.5 is the voltage of half-maximal activation of conductance, and k is the voltage dependence of the activation process (units of millivolts). At room temperature, the net charge (z) moved between closed and open conditions is given by 25.26/k. To evaluate steady-state conductance-voltage relationships, a double Boltzmann that included terms for both activation and block was used:

(2)

where V_0.5 and k correspond to the voltage of half-activation and voltage dependence of activation, respectively, and K_b(0) is the 0 voltage equilibrium constant for the blocking reaction, with z, the fractional charge moved during the blocking reaction, and F, R, and T with their usual meanings. This form of a Boltzmann equation can be related to specific molecular steps for simple activation and blocking schemes in which blockade proceeds strictly from open states, i.e., C O B.

Ensemble averages were generated using our own software. Traces obtained in 0 Ca²⁺ were used to generate an idealized trace that was then subtracted from each record subsequent to averaging of the leak-subtracted records.

Experiments were done at room temperature (21-24°C). All salts and chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Simulation of currents. Simulation of currents was done with custom software developed in the lab, based on manipulation of Q-matrices (Colquhoun and Hawkes, 1981) for up to 30 state models. For the simulations in Figure 11, the following arbitrary model was used:

(Scheme I)

with k₁ = 1000 sec¹, k₁ = 0.5 sec¹_, k_f = 5000 sec¹, and k_r = 2000 sec¹. The block and unblock transitions were voltage-independent while the net charge movement associated with each step along the activation pathway was 2.23e, divided between activation and deactivation steps.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of a BK  subunit variant conferring very rapid inactivation on BK channels

From searches of human EST databases, several cDNA clones of the same gene were identified that show high homology with known BK channel  subunits, KCNMB1 (1) (Knaus et al., 1994), an avian  subunit (Oberst et al., 1997), and KCNMB2 (2) (Wallner et al., 1999; Xia et al., 1999). The identification and isolation of the full-length cDNA clone of the gene h3 was completed through nesting PCR and DNA recombination as described in Materials and Methods. h3 shares extensive homology with other  subunits (Fig. 1). The coding region of 3 is comprised of 771 base pairs and 257 amino acids. The h3 protein shares 24% (62 of 257) identities and 37% (96 of 257) similarities with h1 (191 amino acids). For comparison, Figure 1 also includes sequence for an additional human neuronal  subunit, KCNMB4 (4) (Wickenden et al., 1999; Brenner et al., 2000; Meera et al., 2000; Wallner et al., 2000).

View larger version (72K): [in this window] [in a new window]   Figure 1.   A family of  subunits for the Ca2+-dependent, voltage-gated K+ channel. A, Amino acid alignment of human KCNMB1 1 (h1), quail  (c), human KCNMB2 (h3), human KCNMB3 (h4), and human KCNMB4 (h4) subunits. Highlighted residues are those with identity to aligned residues in at least one other  subunit. The two predicted transmembrane segments (Knaus et al., 1994) are marked with lines both above and below. Residues known to be involved in CTX binding of h1 [amino acids 90-94 of the 1 subunit (Hanner et al., 1997)] are marked with a row of asterisks. B, 5' genomic sequence of KCNMB3 starting with proposed start codon ATG (Riazi et al., 1999). The exon sequence is shaded with the splicing elements in bold. The splicing site is marked by an arrow. C, Standard example of an exon-intron junction and the elements involved in mRNA splicing. The intron sequence is italicized. The 5' and 3' splicing sites are marked by arrows. The genomic sequence for 3 contains several features characteristic of a consensus splice site, including 12 pyrimidines just upstream of the AG junction (bold CTCCCTTTCCCC in B), separated by 2-24 nucleotides from an upstream branch site of seven nucleotides. The subscripts indicate the percent occurrence of the specified base (or type of base) at each consensus position. Py, Pyrimidines; Pu, purines; N, any bases. For mRNA splicing, the intron is defined by a GT-AG rule, which corresponds to the 5' donor and 3' acceptor. Besides the GT-AG, short consensus sequences are required for the completion of splicing, such as a branch site (underlined TCTTTAT in B) located ~18-40 nucleotides upstream of the 3' site (Lewin, 1997).

The nomenclature given in Figure 1 follows current general usage as now defined through GenBank. In a previous publication from this lab (Xia et al., 1999), we used the designation 3 for the then unnamed KCNMB2 subunit, because identification of the avian  subunit (Oberst et al., 1997) preceded identification of KCNMB2 (2). Although the relationship of the avian  subunit to other mammalian  subunits remains to be clarified, the terminology used here follows the GenBank designations for the mammalian  subunit family.

With no signal peptide, the topology analysis suggests that 3 probably shares common structural features with the other  subunits, having two transmembrane domains with both N and C terminals residing intracellularly. The 3 subunit is somewhat unusual in having extended C-terminal sequence. There are two potential N-glycosylation sites (N-X-S/T) in the extracellular loop and one consensus PKC phosphorylation site (Basic_1-3-X_0-2- (S*/T*)-X_0-2-Basic_1-3) located at the penultimate C-terminal residue. 3 shares with the other  subunits four conserved cysteine residues in the extracellular segment.

In the midst of this work, Riazi et al. (1999) published a partial genomic sequence, which, when combined with information from EST data base searches, allowed them to propose a putative gene for a new BK  subunit (Riazi et al., 1999). This putative gene, termed KCNMB3, is identical with our sequence for the 768 base pairs preceding the 3' stop codon (256 amino acids) but differs at the 5' end. The N-terminal amino acid sequence of their putative KCNMB3 is 19 amino acids longer than our 3 beginning from the initial Met. Comparison of their genomic sequence with our cDNA sequence identifies a consensus 3' splicing site in the genomic sequence just at the point of divergence between the two sequences (Fig. 1A). The splicing site is apparently used to generate an initiation ATG site in our cDNA clone and suggests that this 3' splicing site may be used as an alternative splicing site to generate multiple splicing variants (Fig. 1B,C) (Uebele et al., 2000).

It should also be mentioned that, during completion of this work, another group has reported recently on the functional properties of the identical KCNBM3 variant described here (Brenner et al., 2000). However, in this other study, the properties of currents when  + 3 were presumably coexpressed exhibited little difference from currents resulting from the  subunit alone, in marked contrast to the major differences described below.

Tissue distribution of h3 message

Northern blot analysis was used to assess the distribution of 3 mRNA in various human tissues. 3 message was detected in heart, liver, pancreas, adrenal medulla, adrenal cortex, and stomach. Low-level expression of 3 was also detected in skeletal muscle, kidney, thyroid, testis, and small intestine (Fig. 2). The original blot also suggested faint expression in the brain. To determine whether 3 expression might be stronger in particular brain regions, we therefore tested a panel of 14 different brain samples, but only low-level expression of 3 was observed (results not shown). The detected mRNA products are apparently not homogeneous, either within the same tissue or among different tissues. In human heart, as many as four products were detected, 1, 1.8, 2.5, and 6 kb, whereas in other tissues, such as liver, adrenal medulla, and adrenal cortex, only two or three products were visible. However, in human pancreas, four products differing from those of heart were detected, 0.5, 0.9, 1.5, and 2.5. Some of these smaller products (0.5 and 0.9 kb) may be the degradation products of the larger ones. In stomach, the major product was 1.5 kb. This diversity in apparent message size, although possibly indicative of degradation, is also consistent with the possible expression of multiple splice variants of the 3 subunit. Based on our sequence, we would expect transcript sizes on the order of at least 1.0 kb, although with no information about noncoding regions, this is only a minimal estimate. Because of the possibility that multiple splice variants may be present in different tissues, these results do not allow us to identify which tissues express the particular N terminal we describe here.

View larger version (60K): [in this window] [in a new window]   Figure 2.   Distribution of 3 message in various tissues. A, Northern blots show membranes containing human message probed with radiolabeled human 3 sequence. Tissues are as follows: 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas; 9, adrenal medulla; 10, thyroid; 11, adrenal cortex; 12, testis; 13, thymus; 14, small intestine; 15, stomach. The minimal predicted transcript size for the 3 variant described here would be expected to be ~1 kb.

Ca²⁺ and voltage dependence of currents arising from the 3 subunit

The h3 message was coexpressed with Slo  subunits in Xenopus oocytes. Currents obtained from inside-out patches from oocytes expressing h3 and  subunits were both voltage- and Ca²⁺-dependent. Increases in cytosolic Ca²⁺ resulted in shifts in current activation at more negative potentials. In addition, at strong depolarizations and higher Ca²⁺,  + 3 currents exhibited a very rapid, although incomplete, inactivation (Fig. 3). The time constant of the inactivating portion of current was ~0.7-1.5 msec (Fig. 4A). The current inactivation time constant exhibits a moderate dependence on membrane voltage (Fig. 4B) and reaches a Ca²⁺-independent rate at least at the more positive activation potentials (Fig. 4C). Despite the rapid time course of inactivation, inactivation of current is incomplete up to a voltage of +190 mV, and current inactivates to a steady-state level that is dependent on voltage but independent of [Ca²⁺]. The substantial level of sustained current after inactivation at the most positive activation potentials indicates that both the onset of block and recovery from block that underlie the blocking reaction must be rapid. Furthermore, as will be seen in more detail below, there is a range of voltages and [Ca²⁺] over which the rapidity of the inactivation is such that current is reduced, despite the fact that no time-dependent inactivation is discernible in the traces. In such cases, the current does not appear to be inactivating but does exhibit strong rectification. Another interesting aspect of the currents is that, even at moderate Ca²⁺, e.g., 4 µM, at a potential of +60 and more positive, currents rapidly reach their peak value in ~0.5-2 msec.

View larger version (27K): [in this window] [in a new window]   Figure 3.   Coexpression of the 3 subunit with the Slo  subunit produces rapidly inactivating, Ca2+- and voltage-dependent current. A, Traces show currents obtained from an inside-out patch from a Xenopus oocyte injected with cRNA encoding both human 3 and mouse  subunits. Channels were activated by voltage steps from 140 to +180 mV with 0, 1, 4, 10, and 300 µM Ca2+. The voltage protocol is shown at the top. B, Faster time base records of the currents in A are shown for both activation and inactivation and for deactivation. Note that the largest tail current amplitude at 120 mV greatly exceeds the steady-state current level during even the most positive activation steps, indicating that there must be extensive channel unblocking before the peak of the tail current. Also, note that, at 1 and 4 µM Ca2+, the maximal tail current at 120 mV is larger than the maximal peak current activated during the voltage step to +180 mV. This suggests that, at lower Ca2+, many channels become blocked during the rising phase of outward current.

View larger version (27K): [in this window] [in a new window]   Figure 4.   Voltage and Ca2+ dependence of the inactivation time constant of 3 currents. A, Traces on the left show normalized currents (from same patch as Fig. 3) activated with 300 µM Ca2+ for +60, +100, +140, and +180 mV. In each case, the best fit of a single exponential function to the current inactivation time course is plotted over the trace, yielding the time constant of inactivation (i). i was 1.26, 0.77, 0.62, and 0.57 msec for +60, +100, +140, and +180 mV, respectively. Traces on the right show normalized currents (also from Fig. 3) activated at +140 mV for 1, 4, 10, and 300 µM Ca2+. Fitted single exponential functions are also plotted. i was 1.49, 0.74, 0.65, and 0.62 msec for 1, 4, 10, and 300 µM, respectively. B, i is plotted as a function of command potential for four different [Ca2+]. For each Ca2+, there is a small increase in apparent inactivation rate with more depolarized command potential. Each point is the mean ± SEM with four patches for 1 µM Ca2+, 10 patches at 10 µM, nine patches at 300 µM Ca2+, and five patches for 1 mM Ca2+. C, i is replotted as a function of [Ca2+]. At any voltage, there is little indication of any Ca2+ dependence to i at Ca2+ of 10 µM and higher.

To illustrate the unusual features of the 3 currents, Figure 5 compares the properties of currents through channels resulting from the  subunit alone,  + 3, and  + 1, when studied with identical salines and stimulation conditions. Examination of these sets of currents suggests that the 3 subunit, in addition to producing inactivation, shifts the activation range of the resulting channels to more negative potentials at a given Ca²⁺, similar to the 1 subunit. Additionally, the 3 subunit produces a distinct slowing of the deactivation time course at a given Ca²⁺ and voltage, relative to the  subunit alone. However, at Ca²⁺ of 10 µM and higher, this slowing of deactivation is less than observed for the 1 subunit. The inactivation mediated by this 3 subunit differs from inactivation for the previously described 2 subunit in several ways (Wallner et al., 1999; Xia et al., 1999). First, 3 inactivation is much more rapid (~1 vs ~20 msec). Second, there is significant steady-state current during 3 inactivation compared with 2 inactivation. Third, at potentials negative to 0 mV, no time-dependent inactivation is observed for the 3 currents.

View larger version (36K): [in this window] [in a new window]   Figure 5.   Comparison of currents resulting from  alone,  + 3, and  + 1. A, Traces show currents resulting from expression of  alone activated by the indicated voltage protocol with 10, 60, and 300 µM Ca2+. B, Traces show  + 3 currents under the same conditions as in A. C, Traces show currents resulting from  + 1 expression under the same conditions as in A and B, except that the potential both before and after the activation step was 180 mV. The  + 3 combination results in currents that, at a given Ca2+, are activated at more negative potentials than for  subunits alone but somewhat more positive than for  + 1 subunits. This can be most clearly seen in the tail currents. At 60 µM, the step to 120 mV results in only slight current activation for the 3 construct but produces almost 50% activation for the 1 construct.

In the remainder of this paper, we will focus on qualitative aspects of the currents arising from the 3 subunit, including the macroscopic conductance-voltage behavior, pharmacology, and the appearance of single channel openings. Furthermore, we will present results suggesting that an important functional role of the inactivation process may be to produce Ca²⁺-dependent K⁺ current with a relatively fast apparent rate of macroscopic current activation at lower Ca²⁺.

Properties of conductance-voltage curves arising from coexpression of  + 3 subunits

G-V curves were determined in three separate ways (Fig. 6A). First, G-V curves were determined from the tail current amplitude after repolarization from various command potentials. Second, conductances were determined from the peak currents measured during each test step. Third, conductances were determined from the steady-state current remaining at the end of each test step.

View larger version (40K): [in this window] [in a new window]   Figure 6.   The voltage dependence of activation and steady-state properties of inactivation revealed in conductance-voltage curves. A, Examples of G-V curves obtained from tail current amplitude (open triangles), from the peak current (inverted filled triangles) measured during the command step, and from the steady-state current at the end of the voltage step (filled triangles) are shown for one patch bathed with 300 µM Ca2+. For tail currents, each point corresponds to the conductance measured 110 µsec after the nominal imposition of the repolarizing voltage step to 120 mV from a given command potential. Reduction of conductance at the most positive potentials probably reflects some slow cumulative block from contaminating ions. B, A repolarizing step to 160 mV was used to compare tail current amplitude and time course either from just after the peak of outward current during a step to +160 mV or after development of the steady-state current level at +160 mV. Despite the fact that the current near the peak is over twofold larger than the steady-state current, the tail current amplitude in each case is essentially identical. d after repolarization near the peak outward current was 0.70 msec, whereas after repolarization from the steady-state current level it as 0.97 msec. C, Tail current conductances (from the same patch as in A) were determined for 0 (filled circles), 0.5 (open circles), 1 (filled diamonds), 4 (open diamonds), 10 (filled triangles), and 300 (open triangles) µM Ca2+ . The solid lines are single Boltzmann fits (Eq. 1) to the G-V curves. Values were as follows: for 0 µM, V0.5 = 113.3 mV, k = 15.3 mV; for 0.5 µM, V0.5 = 67.1 mV, k = 15.97; for 1 µM, V0.5 = 40.4 mV, k = 15.4 mV; for 4 µM, V0.5 = 16.2 mV, k = 15.9 mV; for 10 µM, V0.5 = 32.2 mV, k = 16.08; for 300 µM, V0.5 = 70.5, k = 17.47 mV. D, Steady-state conductances were determined from the average current level at the end of voltage steps to a given activation potential and plotted as a function of command voltage. Solid lines are fits of the double Boltzmann function given by Equation 2. Values for V0.5 for activation and k for activation were constrained to those obtained in C. E, Peak conductances were calculated from peak currents at each activation potential using a 0 mV reversal potential and plotted as a function of command potential. The lines simply connect the values. F, Voltages at which conductance is half-activated are plotted as a function of [Ca2+] for  alone,  + 1,  + h3, and  + 2(33) in which the 2 inactivation has been deleted. Error bars show SD. For  + 3, points at 0, 10, and 300 µM correspond to 17 patches, points at 1, 4, 1000, and 5000 µM correspond to 6-10 patches, and points at 0.5 µM corresponds to three patches. For  alone, each point corresponds to four patches, for  + 1, six patches, and for  + 2(33), at least five patches for each point. For graphical purposes, points in nominally 0 Ca2+ were plotted at 0.05 µM.

Visual inspection of the G-V curves determined either from the tail currents or the peak currents (Fig. 6A) indicates that a similar maximal conductance can be obtained in both cases. This would seem surprising given the extensive blockade observed in the 3 currents before the repolarizing voltage step. This similarity of the maximal conductance determined from the tail current G-V curves to the maximal conductance in the peak current G-V curves suggests that most channels that are blocked at the end of the activation step must recover essentially instantaneously during the deactivation step. If this is true, a deactivation step at the peak of outward current should elicit the same tail current amplitude as a deactivation step after a steady-state level of inactivation is achieved. This is, in fact, observed (Fig. 6B). Because of this rapid recovery from block, the tail current G-V curves therefore appear to provide a direct indication of the fraction of channels activated by a voltage step to different test potentials, despite the extensive inactivation during the test step. It must be kept in mind, however, that the tail current G-V curves, in fact, represent not only channels that are open before the deactivation step but also channels that were blocked.

Conductances from tail currents were calculated from a repolarizing step to 120 mV after steps to different activation potentials as shown for one patch in Figure 6C. Over all Ca²⁺, a similar absolute maximal conductance is observed from the tail currents, even with 0 Ca²⁺. With elevations in Ca²⁺, the G-V curves show the leftward shift characteristic of BK-type channels. In contrast to the tail current G-V curve, the steady-state conductance (Fig. 6D) exhibits a marked voltage-dependent reduction at more positive potentials, and the absolute amount of steady-state conductance (and current) at all Ca²⁺ from 10 µM and higher is relatively similar at each voltage above 0 mV. Because over these [Ca²⁺] the tail current G-V curves indicate that all channels are essentially maximally activated at any potential above 0 mV (Fig. 6C), this indicates that the blocking reaction is essentially Ca²⁺-independent at 10 µM Ca²⁺ and higher. Even at 1 and 4 µM [Ca²⁺], at sufficiently high voltages, the steady-state conductance also appears to asymptote to values comparable with those at higher Ca²⁺. Thus, qualitatively the blocking process appears to be voltage-dependent but primarily independent of Ca²⁺.

The steady-state conductance was fit in each case with the double Boltzmann given by Equation 2 containing a term for current activation and a second term for a voltage-dependent blocking reaction (Fig. 6D). Both the V_0.5 and k for the activation of conductance were constrained to values obtained from fits to the tail current G-V curves shown in Figure 6C. The resulting double Boltzmann functions then provided an estimate of the voltage dependence of the blocking reaction with values given in the figure legend. The reduction of conductance at each potential positive to 0 mV was essentially identical for all Ca²⁺ of 10 µM and higher. This is the result expected for a Ca²⁺-independent, voltage-dependent blocking process in which the channel activation process is maximally activated over this range of conditions. Results from 17 patches studied with [Ca²⁺] from 10 µM through 5 mM yielded similar conclusions (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Parameters of steady-state block from Equation 2

In the simplest case, Equation 2 arises from the predicted equilibrium condition for a simple three-state model (C O B) in which a voltage-dependent blocking process arises only from open channels. The fitted values for z then provide an indication of the amount of charge movement that occurs during the blocking process. The equivalence of the charge movement during the blocking process over the range of 10 µM to 5 mM Ca²⁺ suggests that, irrespective of the blocking mechanism, the equivalent of a total of ~0.33 elementary charges are moved across the membrane in association with the transition(s) from the open to the blocked condition.

When the identical fitting procedure was used to compare tail current and steady-state G-V curves at 0, 0.5, 1, and 4 µM Ca²⁺, Equation 2 was less successful at accounting for the steady-state currents. Specifically, if the values for the blocking reaction at low [Ca²⁺] were constrained to those obtained from fits to the steady-state G-V curves at higher Ca²⁺, this resulted in large underestimates of the value for maximal conductance at a given Ca²⁺. If, on the other hand, the maximal value of conductance is constrained to be in agreement with the value obtained from fitting the tail current G-V curves, the resulting values for K_b(0) for block and the resulting voltage dependence deviate substantially from those obtained at higher Ca²⁺. Qualitatively, the discrepancy between estimates of the tail current peak conductance and steady-state current peak conductance after accounting for block suggests that, at lower Ca²⁺, more channels are in blocked states than expected based on the expectations of the simple blocking scheme considered here. A more extensive analysis of the blocking mechanism will be presented elsewhere.

In summary, the voltage-dependent reduction of steady-state current observed with the 3 subunit is consistent with the movement of ~0.33 charges moving across the membrane during the transition from fully open states to blocked states. At high Ca²⁺, the steady-state features of block are generally consistent with a simple equilibrium between open and blocked states. At low Ca²⁺, there appears to be more block than might be expected based solely on block of open states alone.

The plot of peak conductance as a function of voltage reveals two other interesting aspects of this current (Fig. 6E). First, at 10 µM Ca²⁺ and above, the peak conductance activated at positive potentials, in general, is similar in most patches to the maximal conductance determined from tail currents, although in some cases the peak conductance never reaches that defined by the tails (Fig. 3). However, for Ca²⁺ well below 10 µM, the maximal conductance activated at positive potentials grossly underestimates the conductance determined from the tail currents. An explanation for this difference is that, at more modest Ca²⁺, the rapid rate of current inactivation relative to a slower rate of current activation reduces the fraction of channels that are open at any time. Thus, when the underlying activation rates of the current are slow, the rapidity of the inactivation process will simply result in a rectifying current. This will be examined more closely below. Second, the relationship between peak conductance and activation voltage exhibits a novel double hump appearance, which is consistently observed in all patches. Such a behavior can, in fact, be predicted from blocking models in which rates of inactivation are rapid relative to rates of activation over a particular range but for which activation exceeds inactivation rates at more positive potentials (C. J. Lingle, unpublished observations).

Because the inactivated channels do, in fact, recover from inactivation extremely rapidly, the tail G-V curves provide a reasonable indication of the Ca²⁺ and voltage dependence of entry of channels into activated states. For comparison with the 3 subunit, we have also examined patches from oocytes expressing  alone,  + 1, or  + 233 [2 with the N-terminal inactivation domain removed (Xia et al., 1999,)]. Figure 6F plots the voltage of half-activation as a function of [Ca²⁺] for all four channel types. The 1 and 2 subunits show the typical marked shift in V_0.5 relative to  alone with less difference at lower Ca²⁺. In contrast,  + 3 produces a marked increase in sensitivity at Ca²⁺ concentrations below 10 µM but produces less of a shift than the 1 subunit at higher Ca²⁺. These results suggest that the 3 subunit may increase the sensitivity of BK channels to submicromolar or low micromolar Ca²⁺ compared with BK channels modulated by other  subunits. Unlike the 1 and 2 subunits, the 3 subunit appears to shift BK gating at low Ca²⁺ as well as higher Ca²⁺, causing a shift of the V_0.5 versus Ca²⁺ curves, which is somewhat parallel to that obtained with  alone.

In summary, the macroscopic  + 3 currents exhibit a number of features that may be important in defining their physiological roles. First, the currents appear more sensitive to lower Ca²⁺ than BK channels formed from other known  subunits. Second, the channels exhibit rapid but incomplete inactivation, such that the macroscopic current exhibits a marked steady-state rectification at positive potentials. Third, outward current can be reduced by the inactivation process even when visible, macroscopic inactivation is not observed (for more details, see Fig. 11). A cautionary remark regarding physiological interpretation of these results is that these measurements were all done in symmetrical K⁺ solutions and at room temperature.

 + 3 channels exhibit unusual single channel openings

A hallmark of BK channels is a characteristic large single channel conductance. To examine the properties of channels arising from  + 3 coexpression, we attempted to identify patches with small numbers of channels. An example of a patch from an oocyte expressing  + 3 subunits is shown in Figure 7. We have been unable to observe single channel openings that are fully resolvable for any such patch studied over a range of Ca²⁺ and activation conditions. Rather, such patches typically exhibit exceedingly brief openings of primarily undefined amplitude, even at a bandwidth of 10 kHz. That such openings do, in fact, represent the behavior of coexpressed  + 3 subunits is supported by the fact that ensemble averages of such openings reveal the characteristic Ca²⁺ and voltage dependence of activation and the rapid but incomplete inactivation of the macroscopic currents (Fig. 7). Furthermore, ensemble variance analysis suggests a single channel conductance in excess of 150 pS at positive activation voltages (data not shown). Because of the rapid flickering behavior of these openings, this value most certainly underestimates the true channel current. This rapid flickering behavior is, in part, consistent with a rapid inactivation process of ~0.5-1.0 msec. However, the failure to resolve any channel openings that approach the expected single channel current level for  subunits alone suggests that other kinetic factors besides the rapid inactivation may contribute to the unusual flickery behavior of the single channels. To date, we have not been successful in identifying a patch with only a single  + 3 channel.

View larger version (43K): [in this window] [in a new window]   Figure 7.   Channel openings resulting from coexpression of  + 3 and exhibiting unusual gating behavior. Traces in each column show channel openings in an inside-out patch bathed with 0, 10, 60, or 300 µM Ca2+, respectively, from left to right. Channel openings were activated by steps to +100 mV (with the voltage protocol shown on the top of the first column). Four consecutive sweeps are shown below each condition. The dotted line indicates a 25 pA current level (250 pS at this voltage). The records were filtered at 10 kHz, and currents were sampled at 100 kHz. Traces on the bottom of each column show ensemble averages for each condition shown above. In each case, at least 60 sweeps were included in each average. Averages exhibit the rapid but incompletely inactivating current seen for the macroscopic currents, despite the fact that no fully resolved channel openings are seen. Single exponential fits to the inactivation time course of the averaged currents yielded values of 0.99, 1.37, and 1.18 msec for 10, 60, and 300 µM Ca2+, respectively, comparable with the inactivation time constants of macroscopic currents. Calibration: 30 pA applies to the individual current sweeps, whereas 15 pA applies to the averaged currents.

Pharmacological properties of the  + 3 channels

In addition to the characteristic shifts in gating with increases in Ca²⁺, BK-type channels exhibit specific pharmacological properties. Because of the unusual properties of the currents resulting from  + 3 coexpression, we examined the pharmacological sensitivity of these currents to various compounds. Extracellular 5 mM 4-aminopyridine was without effect on  + 3 currents. BK currents are typically blocked by extracellular application of tetraethylammonium (TEA) at concentrations less than those affecting other voltage-dependent K⁺ channels. The effect of extracellular TEA on either  alone (Fig. 8A) or  + 3 currents (Fig. 8B) was therefore examined. TEA blocked  + 3 currents with an IC₅₀ of 0.59 ± 0.30 mM at +100 mV (Fig. 8C) compared with an IC₅₀ of 0.43 ± 0.05 mM for block of  alone. Over the range of +20 to +100 mV, the voltage dependence and fitted value for dissociation constant at 0 mV (for values, see legend to Fig. 8D) were similar between the two currents (Fig. 8D).

View larger version (27K): [in this window] [in a new window]   Figure 8.   3 currents exhibit sensitivity to TEA characteristic of other BK currents. A, Traces show currents activated by the indicated voltage-protocol (activation steps from 100 to +100 mV) from an outside-out patch from an oocyte expressing the Slo  subunit alone with 10 µM Ca2+ in the recording pipette. Left panel shows currents in control saline, middle panels show the effects of 1 and 30 mM TEA applied to the extracellular face of the membrane, and the right panel shows currents after removal of TEA. B, Traces show currents activated with the same protocol used in A from an outside-out patch expressing  + 3 subunits. C, The fractional block of current elicited at +100 mV is plotted as a function of extracellular TEA concentration for both  alone (open circles) and  + 3 (filled circles). Fractional block was normalized to the amount of block produced by 30 mM TEA. The IC50 values for block were 0.43 ± 0.05 mM (n = 3) for  with a Hill coefficient of 0.82, and 0.59 ± 0.30 mM (n = 4) for block of  + h3 with a Hill coefficient of 0.94. D, Estimates of the IC50 for block by TEA are plotted as a function of voltage. Fractional block in each case was measured from the steady-state currents at +20 through +100 mV. Solid lines are fits of the function K(V) = K(0) * exp(zFV/RT), where K(0) indicates the IC50 for block by TEA at 0 mV, F, R, and T have their usual meanings, and z is the fitted value for fractional charge moved during the blocking reaction. For , K(0) = 0.18 ± 0.01 mM with z = 0.28 ± 0.025, whereas for  + 3, K(0) = 0.22 ± 0.04 mM, with z = 0.30 ± 0.076.

Extracellular charybdotoxin (CTX) inhibited  + 3 currents with an IC₅₀ of 80.0 ± 10 nM (n = 4) as estimated from the time course of the onset and recovery from CTX blockade at 100 nM (Saito et al., 1997; Xia et al., 1999), assuming that 100% block is defined by 30 mM TEA. This sensitivity of the 3 subunit appears similar to that of the 2 subunit, which is less than for either  + 1 or  alone (Wallner et al., 1999; Xia et al., 1999).

Thus, currents arising from coexpression of  + 3 exhibit pharmacological features characteristic of BK-type channels.

Inactivation results from the 3 N terminus but not the C terminus

Inactivation mediated by the 2 subunit requires an N-terminal structure. Compared with the 2 subunit, the 3 subunit contains a somewhat shorter N terminus but also an extended C terminus that is predicted to be cytosolic. We therefore wished to determine whether either the N- or C-terminal sequences might play a role in inactivation mediated by the 3 subunits. Removal of most of the C terminus of the 3 subunit (construct D3; n > 10 patches) had no discernible affect on the ability of 3 subunits to produce inactivating currents when coexpressed with  (Fig. 9A). In contrast, removal of the N terminus (construct D4; n = 4 patches) resulted in complete removal of inactivation (Fig. 9B).

View larger version (37K): [in this window] [in a new window]   Figure 9.   The 3 N terminus, but not the C terminus, is responsible for inactivation of the  + 3 currents. A, Thirty-five amino acids from the C terminus of the 3 subunit (construct D3) were removed, and the resulting construct was coexpressed with  subunits. Currents retain the typical inactivation behavior of the intact 3 su