Crucial role of a shared extracellular loop in apamin sensitivity and maintenance of pore shape of small- conductance calcium-activated potassium (SK) channels Kate L. Weatheralla, Vincent Seutinb, Jean-François Liégeoisc, and Neil V. Marriona,1 aSchool of Physiology and Pharmacology, University of Bristol, Bristol BS8 1TD, United Kingdom; and Laboratories of bPharmacology and Groupe Interdisciplinaire de Génoprotéomique Appliquée-Neuroscience and cCentre Interfacultaire de Recherche du Médicament, University of Liège, B-4000 Liège, Belgium Edited by Michael D. Cahalan, University of California, Irvine, CA, and approved September 29, 2011 (received for review July 3, 2011) Activation of small-conductance calcium (Ca2+)-dependent potas- apamin does not behave as a classical pore blocker, but blocks sium (KCa2) channels (herein called “SK”) produces membrane hy- using an allosteric mechanism (7). Apamin also interacts with perpolarization to regulate membrane excitability. Three subtypes a serine residue in the S3–S4 extracellular loop to impart a high- (SK1–3) have been cloned and are distributed throughout the ner- sensitivity block of SK2, with mutation of the corresponding vous system, smooth muscle, and heart. It is difficult to discern the threonine in hSK1 to a serine increasing sensitivity of block of physiological role of individual channel subtypes as most blockers hSK1 by the toxin (8). Block of SK channel current by apamin is or enhancers do not discriminate between subtypes. The archetyp- clearly more complicated than these data suggest, as despite ical blocker apamin displays some selectivity between SK channel containing an identical outer pore sequence to the apamin-sen- – subtypes, with SK2 being the most sensitive, followed by SK3 and sitive hSK1 and a serine in this position in the S3 S4 loop, rSK1 – then SK1. Sensitivity of SK1 is species specific, with the human is apamin insensitive. Replacement of the S3 S4 loop of rSK1 isoform being blocked by the toxin, whereas the rat is not. Muta- with that of hSK1 produced a channel with a sensitivity to apa- fi min that was similar to that of hSK1 (8). These data indicate that tion studies have identi ed residues within the outer pore that – suggest apamin blocks by an allosteric mechanism. Apamin also an additional interaction site(s) within the S3 S4 extracellular loop contributes to binding or block by apamin. We have used uses a residue within the S3–S4 extracellular loop to produce PHARMACOLOGY a combination of mutagenesis, functional block, and radioligand a high-sensitivity block. We have identified that a 3-amino acid binding to determine that two amino acids in the S3–S4 loop motif within this loop regulates the shape of the channel pore. affect the shape of the outer and inner pore region of the This motif is required for binding and block by apamin, suggesting channel and are critical for apamin binding and block. Crucially, that a change in pore shape underlies allosteric block. This motif is these findings reveal that due to differences in the S3–S4 loop absent in rat SK1, explaining why it is insensitive to block by sequence, the rat and human isoforms of SK1 have unique pore apamin. The overlapping distribution of SK channel subtype ex- structures that result in a significant difference in their sensitivity pression suggests that native heteromeric channels may be com- both to apamin and to a number of SK channel pore blockers. – mon. We show that the S3 S4 loop of one subunit overlaps the Overlapping distributions of SK channel subunits suggest that outer pore of the adjacent subunit, with apamin interacting with formation of heteromeric channels occurs in vivo (2). Coex- both regions. This arrangement provides a unique binding site for pression of apamin-insensitive rSK1 subunits or a loop mutant of each combination of SK subunits within a coassembled channel rSK2, with apamin-sensitive rSK2 subunits, produces apamin- that may be targeted to produce blockers specific for heteromeric sensitive current. The difference in sensitivity to block of these SK channels. heteromeric channel currents by apamin suggests that the bind- ing site for the toxin is formed by two adjacent subunits, the patch clamp | afterhyperpolarization outer pore region of one and the S3–S4 loop of the adjacent subunit. This proposal is confirmed by producing apamin-sensi- harmacological modulation of small-conductance Ca2+-acti- tive current from coexpression of two apamin-insensitive rSK2 mutant subunits, one with a disrupted S3–S4 loop region and one Pvated potassium (SK/KCa2) channels has been suggested as a potential target for a number of disorders including dementia, with a disrupted outer pore region. We show that the interface depression, and cardiac arrhythmias. Three subtypes of SK chan- between these two regions provides a unique target that will nel have been cloned, with distinct but partially overlapping ex- depend on the SK subtypes contributing to the heteromer. pression patterns (1–3). A nonpeptidic subtype-specific blocker of SK channels has yet to be developed. The lack of SK subtype Results selective blockers combined with this wide distribution means Role of the S3–S4 Extracellular Loop in Binding and Block of SK that their roles cannot be well defined. This lack of specificity Channels by Apamin. Role of single amino acid residues in the binding also means that current available SK blockers have a very narrow and block by apamin of SK channel currents. Alignment of the amino therapeutic window, with an overlap of doses required for acid sequence of the S3–S4 extracellular loop of various SK therapeutic benefit and those producing toxic effects in animal channel subtypes shows a three-amino-acid motif that differs studies (4). between subtypes (Fig. 1). Mutation of serine 245 in rSK2 to SK channels are blocked by the bee venom toxin apamin, mimic the three-amino-acid motif found in hSK1 [rSK2(S245T)] which displays weak subtype selectivity. The toxin is most potent at SK2 (IC50 ∼ 70 pM) (5–9) and SK3 (IC50 ∼ 0.63–6 nM) (7, 9, 10), followed by the human isoform of SK1 (IC50 ∼ 1–8 nM) (8, Author contributions: K.L.W. and N.V.M. designed research; K.L.W., V.S., and J.-F.L. per- 9), whereas the rat isoform of SK1 is apamin insensitive (11, 12). formed research; K.L.W. and J.-F.L. analyzed data; and K.L.W. and N.V.M. wrote Mutation studies have identified a number of residues within the the paper. outer pore that affect block by apamin (7, 8, 13). For example, an The authors declare no conflict of interest. outer pore histidine residue has been shown to be critical for This article is a PNAS Direct Submission. both binding and block by the toxin (7). Structural modeling has 1To whom correspondence should be addressed. E-mail: [email protected]. placed apamin binding with the channel outer pore, interacting This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with this critical histidine residue (7). These data suggested that 1073/pnas.1110724108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1110724108 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Fig. 1. Alignment of the (A)S3–S4 extracellular loop and (B) pore region sequences of SK channel subtypes. Shown is sequence alignment of the S3– S4 loop (A) and pore region (B) of rSK1/mSK1, hSK1, SK2, and SK3. S3–S4 loop regions of rat, human, and mouse isoforms of SK2 and SK3 are iden- tical, as are the pore sequences of human and rat SK1. Amino acid residues within the S3–S4 loop that are mutated are boxed and shown in boldface type. The histidine residue critical for both binding and block by apamin within the outer pore region is boxed and shown in boldface type. The ly- sine/glutamine and glutamate/aspartate residues of the outer pore known to affect apamin sensitivity are boxed and shown in dark gray. The signature selectivity sequence with the pore region for K+ channels is boxed and shown in light gray. produces an inward rectifying current that exhibits a lower sensitivity to block by apamin than wild-type rSK2 (rSK2 IC50, 69.5 ± 13.3 pM, n = 6; rSK2(S245T) IC50, 529 ± 59 pM, n =6,P < 0.0001) (Fig. 2 A and C and Table 1). This shift in apamin sensitivity (∼eightfold) is less than that reported for the opposite mutation (∼19-fold) (10). The lower sensitivity observed here directly results from a reduction in binding affinity for the toxin, with the affinity of binding (KD) to rSK2 being 5.3 ± 1.5 pM (n = 125 4), whereas I-apamin binding to rSK2(S245T) exhibits a KD of 31.3 ± 5.4 pM (n = 3) (Table 1). The corresponding residue in – A Fig. 2. Effect of single mutations within the S3 S4 loop of rSK2 on the rSK1 cannot explain its apamin insensitivity (Fig. 3 ), as this pharmacology of block. (A) Outside-out macropatch currents evoked by subunit possesses a serine residue in this position (Fig. 1). Se- voltage ramps from −100 to +100 mV of expressed rSK2(S245T) channel quence alignment of the S3–S4 loop regions of rSK1 and SK2 subunits in the absence (black) and presence of 300 pM (gray) and 100 nM 246 247 213 214 shows a difference of two amino acids ( YA / LV ), (light gray) apamin. (B) Ramp currents from expressed rSK2(Y246L) channel which may affect the ability of apamin to interact with the subunits in the absence (black) and presence of 300 pM (gray) and 100 nM channel to cause block. Mutation of tyrosine 246 to the corre- (light gray) apamin.