Domain Analysis of the Calcium-activated Potassium Channel SK1 from Rat Brain

Abstract

Two small conductance, calcium-activated potassium channels (SK channels), SK2 and SK3, have been shown to contribute to the afterhyperpolarization (AHP) and to shape the firing behavior in neurons for example in the hippocampal formation, the dorsal vagal nucleus, the subthalamic nucleus, and the cerebellum. In heterologous expression systems, SK2 and SK3 currents are blocked by the bee venom toxin apamin, just as well as the corresponding neuronal AHP currents. However, the functional role and pharmacological profile of SK1 channels from rat brain (rSK1) is still largely unknown, as so far rSK1 homomeric channels could not be functionally expressed. We have performed a domain analysis to elucidate the pharmacological profile and the molecular determinants of rSK1 channel expression by using channel chimeras in combination with immunocytochemistry, immunoblot analysis, and electrophysiology. Our results reveal that the rSK1 subunit is synthesized in cells but does not form functional homomeric channels. Exchanging the carboxyl terminus of rSK1 for that of hSK1 or rSK2 is sufficient to rescue the functional expression of rSK1 channels. Additionally, transplantation of both amino and carboxyl termini of rSK1 onto hSK1 subunits, normally forming functional homomeric channel, hinders their functional expression, while hSK1 channels containing only the rSK1 carboxyl terminus are functional. These results suggest that the lack of functional expression of rSK1 channels is probably due to problems in their assembly and tetramerization but not in their calmodulin-dependent gating. Finally, we show that chimeric channels containing the core domain (S1-S6) of rSK1, unlike hSK1, are apamin-insensitive.Neurons encode information in the form of spike frequency. Spike frequency adaptation (SFA) 1 denotes the progressive decrease in firing frequency in response to prolonged depolarizations (1). SFA is mainly due to the activation of ion channels generating the afterhyperpolarizations (AHPs) that follow action potentials. The more pronounced the AHPs, the less the neuron fires and the stronger is SFA (1). SFA and the underlying AHP show two temporally distinct phases in cortical neurons (2). The medium AHP (mAHP), lasting 100 -300 ms, can be observed after single or bursts of action potentials (3, 4). Different K ϩ currents underlie the mAHP, among which a prominent role is played by the Ca 2ϩ -activated K ϩ (K Ca ) current I AHP (4). I AHP is activated by Ca 2ϩ entering the neurons during action potentials and hyperpolarizes the membrane, thereby generating the early phase of SFA and regulating the tonic firing frequency of neurons (4). This current is selectively blocked by the toxins apamin, scyllatoxin, and tamapin (4, 5). The slow AHP activates and deactivates around 10-fold slower compared with the mAHP. sAHP is also mediated by a K Ca current termed sI AHP , which is not blocked by apamin or any other known K ϩ channel blockers in brain slices (2). The suppression of sI AHP by a number of neurotransmitters leads to a dra...

Crucial role of a shared extracellular loop in apamin sensitivity and maintenance of pore shape of small-conductance calcium-activated potassium (SK) channels

“…The three-amino-acid motif in the S3-S4 loop of SK channels (SLV in rSK1, TYA in hSK1, SYA in SK2, and SYT in SK3) is an essential molecular determinant for apamin sensitivity. These data resolve the dichotomy that rSK1 is insensitive to apamin (11,12), yet has the same pore sequence as the apamin-sensitive hSK1. It is because rSK1 displays SLV in the S3-S4 loop that prevents binding of apamin.…”

“…Amino acids 246 YA 247 / 213 LV 214 dictate apamin binding and block. Expression of homomeric rSK1 subunits does not produce functional channels (11,12,15). Functional channel current is produced on expression of a chimeric channel (rSK1*), where the carboxyl terminus of rSK1 is replaced by that from rSK2 (11).…”

“…SK channels are blocked by the bee venom toxin apamin, which displays weak subtype selectivity. The toxin is most potent at SK2 (IC 50 ∼ 70 pM) (5-9) and SK3 (IC 50 ∼ 0.63-6 nM) (7, 9, 10), followed by the human isoform of SK1 (IC 50 ∼ 1-8 nM) (8, 9), whereas the rat isoform of SK1 is apamin insensitive (11,12). Mutation studies have identified a number of residues within the outer pore that affect block by apamin (7,8,13).…”

CaV1.3-Driven SK Channel Activation Regulates Pacemaking and Spike Frequency Adaptation in Mouse Chromaffin Cells

“…All three KCNN ␣-subunits were found to be expressed in MCCs, giving rise to a bisigmoidal dose-response curve of apamin on slow tail currents. Different SK channel isoforms are characterized by distinct apamin sensitivities, with SK2 being most sensitive (IC 50 ϭ 60 -70 pM), SK3 showing an intermediate sensitivity (IC 50 ϭ 0.63-6 nM), and rat/mouse SK1 being rather insensitive (Ishii et al, 1997;Grunnet et al, 2001;Benton et al, 2003;D'hoedt et al, 2004;Weatherall et al, 2011). Heterologous expression studies (Ishii et al, 1997) have shown that SK channels might be heteromeric in nature, giving rise to channels with intermediate apamin sensitivities.…”

A Functional Role for Small-Conductance Calcium-Activated Potassium Channels in Sensory Pathways Including Nociceptive Processes

“…IK channels are insensitive to these blockers. In the rat, it seems likely that SK1 subunits cannot form functional channels by themselves (Benton et al, 2003;D'hoedt et al, 2004;Monaghan et al, 2004). However, they can form functional channels in combination with SK2 (Benton et al, 2003).…”