AbstractPancreatic β-cells have the unique ability to couple glucose metabolism to insulin secretion. This capacity is generally attributed to the ability of ATP to inhibit KATPchannels, and the consequent β-cell membrane depolarization and excitation. This notion has recently been challenged by a study which demonstrated that high glucose (HG) downregulates the cell surface KATPchannels, and thereby leads to β-cell depolarisation and excitation. The authors attributed the downregulation to HG-induced protein kinase C (PKC) activation and the consequent increase in channel endocytosis. This interpretation, however, is inconsistent with our previous findings that PKC activation does not affect endocytosis. To address this controversy, we revisited the problem: we have used cell biological and electrophysiological approaches combined with the pharmacological activator of PKC, PMA (phorbol 12-myristate 13-acetate). We first confirm that PKC does not play a role in KATPchannel endocytosis; instead, it downregulates the channel by promoting lysosomal degradation coupled with reduced recycling. We then show that (i) mutation of the dileucine motif (355LL356) in the C-terminal domain of the Kir6.2 subunit of the KATPchannel complex prevents lysosomal degradation; (ii) lysosomal targeting is mediated by the EHD (Eps15 homology domain– containing) proteins; and (iii) the PKC isoform responsible for channel degradation is PKCε. Taken together with the published data, we suggest that HG promotes β-cell excitability via two mechanisms: ATP-dependent channel inhibition and ATP-independent, PKCε-dependent channel degradation. The results likely have implications for glucose induced biphasic insulin secretion.

Slo3 is a pH-sensitive and weakly voltage-sensitive potassium channel that is essential for male fertility in mouse and whose expression is regarded as sperm-specific. These properties have proposed Slo3 as a candidate target for male contraceptive drugs. Nonetheless, the tissue distribution of Slo3 expression has not been rigorously studied yet. Applying computational and RT-PCR approaches, we identified expression of two short Slo3 isoforms in somatic mouse tissues such as brain, kidney and eye. These isoforms, which seem to result of transcription starting sites between exons 20 and 21, have an identical open reading frame, both encoding the terminal 381 amino acids of the cytosolic Slo3 domain. We corroborated the expression of these isoforms in mouse brain and testis by Western-blot. The complete isoform encoding the Slo3 ion channel was uniquely detected in testis, both at transcript and protein level. Although the functional role of the cytosolic Slo3 isoforms remains to be established, we propose that they may have a functional effect by modulating Slo channels trafficking and/or activity. This study confirms that expression of full-length Slo3 is sperm-specific but warns against developing contraceptive drugs targeting the C-terminal tail of Slo3 channels.

Background and PurposeThe Slo3 (K(Ca)5.1) channel is a major component of mammalian KSper (sperm potassium conductance) channels and inhibition of these channels by quinine and barium alters sperm motility. The aim of this investigation was to determine the mechanism by which these drugs inhibit Slo3 channels.Experimental ApproachMouse (m) Slo3 (K(Ca)5.1) channels or mutant forms were expressed in Xenopus oocytes and currents recorded with 2-electrode voltage-clamp. Gain-of-function mSlo3 mutations were used to explore the state-dependence of the inhibition. The interaction between quinidine and mSlo3 channels was modelled by in silico docking.Key ResultsSeveral drugs known to block KSper also affected mSlo3 channels with similar levels of inhibition. The inhibition induced by extracellular barium was prevented by increasing the extracellular potassium concentration. R196Q and F304Y mutations in the mSlo3 voltage sensor and pore, respectively, both increased channel activity. The F304Y mutation did not alter the effects of barium, but increased the potency of inhibition by both quinine and quinidine approximately 10-fold; this effect was not observed with the R196Q mutation.Conclusions and ImplicationsBlock of mSlo3 channels by quinine, quinidine and barium is not state-dependent. Barium inhibits mSlo3 outside the cell by interacting with the selectivity filter, whereas quinine and quinidine act from the inside, by binding in a hydrophobic pocket formed by the S6 segment of each subunit. Furthermore, we propose that the Slo3 channel activation gate lies deep within the pore between F304 in the S6 segment and the selectivity filter.

Members of the six-transmembrane segment family of ion channels share a common structural design. However, there are sequence differences between the members that confer distinct biophysical properties on individual channels. Currently, we do not have 3D structures for all members of the family to help explain the molecular basis for the differences in their biophysical properties and pharmacology. This is due to low-level expression of many members in native or heterologous systems. One exception is rat Kv1.2 which has been overexpressed in Pichia pastoris and crystallised. Here, we tested chimaeras of rat Kv1.2 with the hERG channel for function in Xenopus oocytes and for overexpression in Pichia . Chimaera containing the S1–S6 transmembrane region of HERG showed functional and pharmacological properties similar to hERG and could be overexpressed and purified from Pichia . Our results demonstrate that rat Kv1.2 could serve as a surrogate to express difficult-to-overexpress members of the six-transmembrane segment channel family.

The patch clamp technique revolutionized the study of ion channels and is considered the gold standard of measuring ion channel activity, from the academic laboratory to industrial-scale drug screening. This technique enables the study of ion channels, from single molecules up to the whole-cell ion channel population, and in their native environment. Whilst the study of single protein molecular behavior is the ultimate goal of biophysicists from all fields, this is a routine ability for ion channel specialists. This chapter is aimed at helping the beginner to design patch clamp experiments and to obtain the fundamental micropipette configurations with mammalian cells: cell-attached patch, whole cell, inside-out patch, and outside-out patch.

N-methyl-d-aspartate receptors (NMDARs) display differences in their sensitivity to the channel blockers Mg(2+) and memantine that are dependent on the identity of the NR2 subunit present in the receptor-channel complex. This study used two-electrode voltage-clamp recordings from Xenopus laevis oocytes expressing recombinant NMDARs to investigate the actions of Mg(2+) and memantine at the two NMDARs displaying the largest differences in sensitivity to these blockers, namely NR1/NR2A and NR1/NR2D NMDARs. In addition, NR2A/2D chimeric subunits have been employed to examine the effects of pore-forming elements and ligand-binding domains (LBD) on the potency of the block produced by each of these inhibitors. Our results show that, as previously documented, NR2D-containing NMDARs are less sensitive to voltage-dependent Mg(2+) block than their NR2A-containing counterparts. The reduced sensitivity is determined by the M1M2M3 membrane-associated regions, as replacing these regions in NR2A subunits with those found in NR2D subunits results in a approximately 10-fold reduction in Mg(2+) potency. Intriguingly, replacing the NR2A LBD with that from NR2D subunits results in a approximately 2-fold increase in Mg(2+) potency. Moreover, when responses mediated by NR1/NR2A NMDARs are evoked by the partial agonist homoquinolinate, rather than glutamate, Mg(2+) also displays an increased potency. Memantine block of glutamate-evoked currents is most potent at NR1/NR2D NMDARs, but no differences are observed in its ability to inhibit NR2A-containing or NR2A/2D chimeric NMDARs. We suggest that the potency of block of NMDARs by Mg(2+) is influenced not only by pore-forming regions but also the LBD and the resulting conformational changes that occur following agonist binding.

Large-conductance-Ca2+-activated potassium Slo1 channels are well characterised members of the Slo channel family. The other members, Slo3 and Slo2.χ, are less characterised and their physiological roles are elusive. Little is known of the pH-sensitive Slo3 channel, aside from a role regulating Ca2+ influx through CatSper channels during capacitation in spermatozoa. This thesis aims to investigate mSlo3 pharmacology and determine if mSlo3- EST’s encode novel isoforms. Na+-activated Slo2.χ channels (Slo2.1/Slo2.2) are thought to mediate adaptation of neuronal action potential firing rate and slow after hyperpolarisations following repetitive firing. We aim to characterise hSlo2.2a currents and their bithionol sensitivity, an activator of a rat isoform. Mutation of the conserved phenylalanine in the S6-domain (mSlo3-F304/hSlo1-F380) to tyrosine has similar effects on Slo channels, namely a negative shift in the voltagedependence of activation and an increase in membrane conductance. mSlo3 pharmacology matches the IKSper profile and suggests roles for mSlo3 as a pharmacological target in infertility treatment. This work identified a novel splice-variant of mSlo3; CV562866 lacks transmembrane domains and is more widely expressed than mSlo3. CV562866 protein is expressed in vivo, and contains the nominal RCK2 domain. CV562866 affects functional expression of mSlo3 and mSlo1 channels in Xenopus oocytes, decreasing and increasing expression respectively. This study examined the hSlo2.2a channel characteristics, which unlike the previously reported rSlo2.2a channel, mediates a non-rectifying K+ current. Bithionol activates hSlo2.2a channels with an EC50 of 1.29±0.12μM and activation was Na+ co-operative. We found a candidate bithionol binding site in the C-terminal domain near the “Cl- bowl”. Due to toxicity of hSlo2.1 expression; determination of the bithionol selectivity of Slo2.χ channels was unattainable. In vivo, Slo2.χ channels act as a background K+ conductance, activated by a variety of conditions, stabilising the resting membrane potential of the cell. This study suggests Slo2.χ are viable pharmacological targets for treatment of inflammatory pain.