Jiuping Ding

Protein Kinase Activation Increases Insulin Secretion by Sensitizing the Secretory Machinery to Ca2

Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca2+, we characterize three kinetically different pools of vesicles in rat pancreatic β-cells, namely, a highly calcium-sensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is ∼20 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca2+, we find that the HCSP is a distinct population of vesicles from those colocalized with Ca2+ channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca2+.

Hg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels from First Scorpion Kunitz-type Potassium Channel Toxin Family

Background: The potassium channel inhibitory activity of scorpion Kunitz-type toxins has not yet been determined. Results: We identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. Conclusion: A novel peptide, Hg1, specific for Kv1.3 channel, was found. Significance: Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases. The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nm for recombinant LmKTT-1a, 160 nm for LmKTT-1b, 124 nm for LmKTT-1c, 136 nm for BmKTT-1, 420 nm for BmKTT-2, 760 nm for BmKTT-3, and 107 nm for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ∼50–80% of Kv1.3 channel currents at a concentration of 1 μm. The exception was rBmKTT-3, which had weak activity. The IC50 values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ∼129.7, 371.3, and 6.2 nm, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.

Characterization of voltage-and Ca2+-activated K+ channels in rat dorsal root ganglion neurons†

Auxiliary β‐subunits associated with pore‐forming Slo1 α‐subunits play an essential role in regulating functional properties of large‐conductance, voltage‐ and Ca2+‐activated K+ channels commonly termed BK channels. Even though both noninactivating and inactivating BK channels are thought to be regulated by β‐subunits (β1, β2, β3, or β4), the molecular determinants underlying inactivating BK channels in native cells have not been extensively demonstrated. In this study, rβ2 (but not rβ3‐subunit) was identified as a molecular component in rat lumbar L4‐6 dorsal root ganglia (DRG) by RT‐PCR responsible for inactivating large‐conductance Ca2+‐dependent K+ currents (BKi currents) in small sensory neurons. The properties of native BKi currents obtained from both whole‐cell and inside‐out patches are very similar to inactivating BK channels produced by co‐expressing mSlo1 α‐ and hβ2‐subunits in Xenopus oocytes. Intracellular application of 0.5 mg/ml trypsin removes inactivation of BKi channels, and the specific blockers of BK channels, charybdotoxin (ChTX) and iberiotoxin (IbTX), inhibit these BKi currents. Single BKi channel currents derived from inside‐out patches revealed that one BKi channel contained three rβ2‐subunits (on average), with a single‐channel conductance about 217 pS under 160 K+ symmetrical recording conditions. Blockade of BKi channels by 100 nM IbTX augmented firing frequency, broadened action potential waveform and reduced after‐hyperpolarization. We propose that the BKi channels in small diameter DRG sensory neurons might play an important role in regulating nociceptive input to the central nervous system (CNS). J. Cell. Physiol. 212: 348–357, 2007.

Venomic and transcriptomic analysis of centipede Scolopendra subspinipes dehaani.

Centipedes have venom glands in their first pair of limbs, and their venoms contain a large number of components with different biochemical and pharmacological properties. However, information about the compositions and functions of their venoms is largely unknown. In this study, Scolopendra subspinipes dehaani venoms were systematically investigated by transcriptomic and proteomic analysis coupled with biological function assays. After random screening approximately 1500 independent clones, 1122 full length cDNA sequences, which encode 543 different proteins, were cloned from a constructed cDNA library using a pair of venom glands from a single centipede species. Neurotoxins, ion channel acting components and venom allergens were the main fractions of the crude venom as revealed by transcriptomic analysis. Meanwhile, 40 proteins/peptides were purified and characterized from crude venom of S. subspinipes dehaani. The N-terminal amino acid sequencing and mass spectrum results of 29 out of these 40 proteins or peptides matched well with their corresponding cDNAs. The purified proteins/peptides showed different pharmacological properties, including the following: (1) platelet aggregating activity; (2) anticoagulant activity; (3) phospholipase A(2) activity; (4) trypsin inhibiting activity; (5) voltage-gated potassium channel activities; (6) voltage-gated sodium channel activities; (7) voltage-gated calcium channel activities. Most of them showed no significant similarity to other protein sequences deposited in the known public database. This work provides the largest number of protein or peptide candidates with medical-pharmaceutical significance and reveals the toxin nature of centipede S. subspinipes dehaani venom.

Activation of Large-Conductance Calcium-Activated Potassium Channels by Puerarin: The Underlying Mechanism of Puerarin-Mediated Vasodilation

Puerarin is the main isoflavone found in Pueraria lobata (Willd) Ohwi, which has been used in therapy for various cardiovascular diseases. The present study examined the effects of puerarin on the large-conductance voltage- and Ca2+-activated potassium (BKCa) channel and on rat thoracic aortas. BKCa channels encoded with either α (BK-α) or α/β subunits (BK-α+β1) were heterologously expressed in Xenopus oocytes or human embryonic kidney 293 cells. The activities of BKCa channels were measured using excised patch-clamp recordings. Puerarin activated BK-α+β1 currents with a half-maximal concentration (EC50) of 0.8 nM and a Hill coefficient of 1.11 at 10 μMCa2+ and with an EC50 of 12.6 nM and a Hill coefficient of 1.08 at 0 μMCa2+. Puerarin (1 nM) induced a 16-mV leftward shift in the conductance-voltage curve for BK-α+β1 currents at 10 μMCa2+ and at 100 nM induced a 26-mV leftward shift at 0 μMCa2+. Puerarin mainly increased the BK-α+β1 channel open probability without changing the unitary conductance. Activation was also detected in the absence of the β1 subunit. A deglycosylated analog of puerarin, daidzein, also activated BKCa channels with weaker potency. In addition, puerarin (0.1 to 1000 μM) caused concentration-dependent relaxations of rat thoracic aortic rings contracted with 1 μM noradrenaline bitartrate (EC50 = 1.1 μM). These were significantly inhibited by 50 nM iberiotoxin, a specific blocker of BKCa channels. This is the first study demonstrating that puerarin activates BKCa channels, especially BK-α+β1 channels. The activation of the BKCa channel probably contributes to the puerarin-mediated vasodilation action.

BmP09, a "long chain" scorpion peptide blocker of BK channels

A novel “long chain” toxin BmP09 has been purified and characterized from the venom of the Chinese scorpion Buthus martensi Karsch. The toxin BmP09 is composed of 66 amino acid residues, including eight cysteines, with a mass of 7721.0 Da. Compared with the B. martensi Karsch AS-1 as a Na+ channel blocker (7704.8 Da), the BmP09 has an exclusive difference in sequence by an oxidative modification at the C terminus. The sulfoxide Met-66 at the C terminus brought the peptide a dramatic switch from a Na+ channel blocker toaK+ channel blocker. Upon probing the targets of the toxin BmP09 on the isolated mouse adrenal medulla chromaffin cells, where a variety of ion channels coexists, we found that the toxin BmP09 specifically blocked large conductance Ca2+- and voltage-dependent K+ channels (BK) but not Na+ channels at a range of 100 nm concentration. This was further confirmed by blocking directly the BK channels encoded with mSlo1 α-subunits in Xenopus oocytes. The half-maximum concentration EC50 of BmP09 was 27 nm, and the Hill coefficient was 1.8. In outside-out patches, the 100 nm BmP09 reduced ∼70% currents of BK channels without affecting the single-channel conductance. In comparison with the “short chain” scorpion peptide toxins such as Charybdotoxin, the toxin BmP09 behaves much better in specificity and reversibility, and thus it will be a more efficient tool for studying BK channels. A three-dimensional simulation between a BmP09 toxin and an mSlo channel shows that the Lys-41 in BmP09 lies at the center of the interface and plugs into the entrance of the channel pore. The stable binding between the toxin BmP09 and the BK channel is favored by aromatic π -π interactions around the center.

Structural Basis for Toxin Resistance of β4-Associated Calcium-activated Potassium (BK) Channels

The functional diversity of large conductance Ca2+- and voltage-dependent K+ (BK) channels arises mainly from co-assembly of the pore-forming mSlo α subunits with four tissue-enriched auxiliary β subunits. The structural basis of the interaction between α subunits with β subunits is not well understood. Using computational and experimental methods, we demonstrated that four mSlo turrets decentralized distally from the channel pore to provide a wide open conformation and that the mSlo and hβ4 subunits together formed a “helmet” containing three basic residues (Lys-120, Arg-121, and Lys-125), which impeded the entry of charybdotoxin (ChTX) by both the electrostatic interaction and limited space. In addition, the tyrosine insert mutant (in100Y) showed 56% inhibition, with a Kd = 17 nm, suggesting that the hβ4 lacks an external ChTX-binding site (Tyr-100). We also found that mSlo had an internal binding site (Tyr-294) in the α subunits that could “permanently” block 15% of mSlo+hβ4 currents in the presence of 100 nm ChTX. These findings provide a better understanding of the diverse interactions between α and β subunits and will improve the design of channel inhibitors.

Structural and Functional Diversity of Acidic Scorpion Potassium Channel Toxins

Background Although the basic scorpion K+ channel toxins (KTxs) are well-known pharmacological tools and potential drug candidates, characterization the acidic KTxs still has the great significance for their potential selectivity towards different K+ channel subtypes. Unfortunately, research on the acidic KTxs has been ignored for several years and progressed slowly. Principal Findings Here, we describe the identification of nine new acidic KTxs by cDNA cloning and bioinformatic analyses. Seven of these toxins belong to three new α-KTx subfamilies (α-KTx28, α-KTx29, and α-KTx30), and two are new members of the known κ-KTx2 subfamily. ImKTx104 containing three disulfide bridges, the first member of the α-KTx28 subfamily, has a low sequence homology with other known KTxs, and its NMR structure suggests ImKTx104 adopts a modified cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif that has no apparent α-helixs and β-sheets, but still stabilized by three disulfide bridges. These newly described acidic KTxs exhibit differential pharmacological effects on potassium channels. Acidic scorpion toxin ImKTx104 was the first peptide inhibitor found to affect KCNQ1 channel, which is insensitive to the basic KTxs and is strongly associated with human cardiac abnormalities. ImKTx104 selectively inhibited KCNQ1 channel with a Kd of 11.69 µM, but was less effective against the basic KTxs-sensitive potassium channels. In addition to the ImKTx104 toxin, HeTx204 peptide, containing a cystine-stabilized α-helix-loop-helix (CS-α/α) fold scaffold motif, blocked both Kv1.3 and KCNQ1 channels. StKTx23 toxin, with a cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif, could inhibit Kv1.3 channel, but not the KCNQ1 channel. Conclusions/Significance These findings characterize the structural and functional diversity of acidic KTxs, and could accelerate the development and clinical use of acidic KTxs as pharmacological tools and potential drugs.

Differential Regulation of Action Potentials by Inactivating and Noninactivating BK Channels in Rat Adrenal Chromaffin Cells

Large-conductance Ca(2+)-activated K(+) (BK) channels can regulate cellular excitability in complex ways because they are able to respond independently to two distinct cellular signals, cytosolic Ca(2+) and membrane potential. In rat chromaffin cells (RCC), inactivating BK(i) and noninactivating (BK(s)) channels differentially contribute to RCC action potential (AP) firing behavior. However, the basis for these differential effects has not been fully established. Here, we have simulated RCC action potential behavior, using Markovian models of BK(i) and BK(s) current and other RCC currents. The analysis shows that BK current influences both fast hyperpolarization and afterhyperpolarization of single APs and that, consistent with experimental observations, BK(i) current facilitates repetitive firing of APs, whereas BK(s) current does not. However, the key functional difference between BK(i) and BK(s) current that accounts for the differential firing is not inactivation but the more negatively shifted activation range for BK(i) current at a given [Ca(2+)].

Intersubunit Coupling in the Pore of BK Channels

The structural basis underlying the gating of large conductance Ca2+-activated K+ (BK) channels remains elusive. We found that substitution of Leu-312 in the S6 transmembrane segment of mSlo1 BK channels with hydrophilic amino acids of smaller side-chain volume favored the open state. The sensitivities of channels to calcium and voltage were modified by some mutations and completely abolished by others. Interpretation of the results in terms of an allosteric model suggests that the calcium-insensitive mutants greatly destabilize the closed relative to the open conformation and may also disrupt the allosteric coupling between Ca2+ or voltage sensors and the gate. Some Phe-315 mutations also favor the open state, suggesting that Leu-312 and Phe-315 may interact in the closed state, forming a major energy barrier that the channel has to overcome to open. Homology modeling and molecular dynamic simulations further support that the side chain of Leu-312 can couple strongly with the aromatic ring of Phe-315 in neighboring subunits (L-F coupling) to maintain the channel closed. Additionally, single-channel recordings indicate that the calcium-insensitive mutants, whose kinetics can be approximately characterized by a two-state closed-open (C-O) model, exhibit nearly 100% open probability under physiological conditions without alterations in single-channel conductance. These findings provide a basis for understanding the structure and gating of the BK channel pore.