Zhe Zhang

Virginia Commonwealth University, Richmond, VA, United States

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Publications (20)89.89 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca2+). Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca2+-driven gating of BK channels but a molecular understanding of this interplay or even of the PIP2 regulation of this channel's activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca2+ and PIP2 in regulating BK channel activity. In the absence of Ca2+, RCK1 structural elements limit channel activation through a decrease in the channel's PIP2 apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that a) connect either the Ca2+ coordination site(D367 in the KDRDD loop) or its flanking basic residues to PIP2 interacting residues (K392 and R393) found in the αB helix or b) are involved in hydrophobic interactions between αA and αB helix of the RCK1 domain. In the presence of Ca2+, the RCK1 inhibitory influence of channel-PIP2 interactions and channel activity is relieved by Ca2+ engaging D367. Our results demonstrate that along with Ca2+ and voltage, PIP2 is a third factor critical to the integral control of BK channel activity.
    Journal of Biological Chemistry 04/2014; · 4.65 Impact Factor
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    ABSTRACT: Slo channels are large conductance K (+) channels that display marked differences in their gating by intracellular ions. Among them, the Slo1 and C. elegans SLO-2 channels are gated by calcium (Ca ( 2+) ), while mammalian Slo2 channels are activated by both sodium (Na (+) ) and chloride (Cl (-) ). Here, we report that SLO-2 channels, SLO-2a and a novel N-terminal variant isoform, SLO-2b, are activated by Ca ( 2+) and voltage, but in contrast to previous reports they do not exhibit Cl (-) sensitivity. Most importantly, SLO-2 provides a unique case in the Slo family for sensing Ca ( 2+) with the high-affinity Ca ( 2+) regulatory site in the RCK1 but not the RCK2 domain, formed through interactions with residues E319 and E487 (that correspond to D362 and E535 of Slo1, respectively). The SLO-2 RCK2 domain lacks the Ca ( 2+) bowl structure and shows minimal Ca ( 2+) dependence. In addition, in contrast to SLO-1, SLO-2 loss-of-function mutants confer resistance to hypoxia in C. elegans. Thus, the C. elegans SLO-2 channels possess unique biophysical and functional properties.
    Channels (Austin, Tex.) 04/2013; 7(3). · 1.91 Impact Factor
  • Biophysical Journal 01/2013; 104(2):357-. · 3.67 Impact Factor
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    ABSTRACT: The family C G protein-coupled receptor (GPCR) T1R2 and T1R3 heterodimer functions as a broadly acting sweet taste receptor. Perception of sweet taste is a species-dependent physiological process. It has been widely reported that New World monkeys and rodents are not able to perceive some of the artificial sweeteners and sweet-tasting proteins that can be perceived by humans, apes, and Old World monkeys. Until now, only the sweet receptors of humans, mice and rats have been functionally characterized. Here we report characterization of the sweet taste receptor (T1R2/T1R3) from a species of New World primate, squirrel monkey. Our results show that the heterodimeric receptor of squirrel monkey does not respond to artificial sweeteners aspartame, neotame, cyclamate, saccharin and sweet-tasting protein monellin, but surprisingly, it does respond to thaumatin at high concentrations (>18 μM). This is the first report demonstrating that species of New World monkey can perceive some specific sweet-tasting proteins. Furthermore, the sweet receptor of squirrel monkey responses to the such sweeteners cannot be inhibited by the sweet inhibitor lactisole. We compared the response differences of the squirrel monkey and human receptors and found that the residues in T1R2 determine species-dependent sweet taste toward saccharin, while the residues in either T1R2 or T1R3 are responsible for the sweet taste difference between humans and squirrel monkeys toward monellin. Molecular models indicated that electrostatic properties of the receptors probably mediate the species-dependent response to sweet-tasting proteins.
    Biochemical and Biophysical Research Communications 10/2012; 427(2):431–437. · 2.41 Impact Factor
  • Biophysical Journal 01/2012; 102(3):301-. · 3.67 Impact Factor
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    ABSTRACT: Hypercholesterolemia is a well-known risk factor for cardiovascular disease. In the heart, activation of K(ACh) mediates the vagal (parasympathetic) negative chronotropic effect on heart rate. Yet, the effect of cholesterol on K(ACh) is unknown. Here we show that cholesterol plays a critical role in modulating K(ACh) currents (I(K,ACh)) in atrial cardiomyocytes. Specifically, cholesterol enrichment of rabbit atrial cardiomyocytes led to enhanced channel activity while cholesterol depletion suppressed I(K,ACh). Moreover, a high-cholesterol diet resulted in up to 3-fold increase in I(K,ACh) in rodents. In accordance, elevated currents were observed in Xenopus oocytes expressing the Kir3.1/Kir3.4 heteromer that underlies I(K,ACh). Furthermore, our data suggest that cholesterol affects I(K,ACh) via a mechanism which is independent of both PI(4,5)P(2) and Gβγ. Interestingly, the effect of cholesterol on I(K,ACh) is opposite to its effect on I(K1) in atrial myocytes. The latter are suppressed by cholesterol enrichment and by high-cholesterol diet, and facilitated following cholesterol depletion. These findings establish that cholesterol plays a critical role in modulating I(K,ACh) in atrial cardiomyocytes via a mechanism independent of the channel's major modulators.
    Journal of Biological Chemistry 12/2011; 287(7):4925-35. · 4.65 Impact Factor
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    ABSTRACT: The heterodimer of Tas1R2 and Tas1R3 is a broadly acting sweet taste receptor, which mediates mammalian sweet taste toward natural and artificial sweeteners and sweet-tasting proteins. Perception of sweet taste is a species-selective physiological process. For instance, artificial sweeteners aspartame and neotame taste sweet to humans, apes, and Old World monkeys but not to New World monkeys and rodents. Although specific regions determining the activation of the receptors by these sweeteners have been identified, the molecular mechanism of species-dependent sweet taste remains elusive. Using human/squirrel monkey chimeras, mutagenesis, and molecular modeling, we reveal that the different responses of mammalian species toward the artificial sweeteners aspartame and neotame are determined by the steric effect of a combination of a few residues in the ligand binding pocket. Residues S40 and D142 in the human Tas1R2, which correspond to residues T40 and E142 in the squirrel monkey Tas1R2, were found to be the critical residues for the species-dependent difference in sweet taste. In addition, human Tas1R2 residue I67, which corresponds to S67 in squirrel monkey receptor, modulates the higher affinity of neotame than of aspartame. Our studies not only shed light on the molecular mechanism of species-dependent sweet taste toward artificial sweeteners, but also provide guidance for designing novel effective artificial sweet compounds.
    Journal of Neuroscience 07/2011; 31(30):11070-6. · 6.91 Impact Factor
  • Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
  • Biophysical Journal - BIOPHYS J. 01/2011; 100(3).
  • Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
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    ABSTRACT: Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.
    Journal of Biological Chemistry 10/2010; 285(51):39790-800. · 4.65 Impact Factor
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    ABSTRACT: Slo2 Na(+)-activated potassium channels are widely expressed in neurons and other cells, such as kidney, heart, and skeletal muscle. Although their important physiological roles continue to be appreciated, molecular determinants responsible for sensing intracellular Na(+) remain unknown. Here we report identification of an Na(+) regulatory site, similar to an Na(+) coordination motif described in Kir channels, localized in the RCK2 domain of Slo2.2 channels. Molecular simulations of the homology-modeled Slo2.2 RCK2 domain provided structural insights into the organization of this Na(+) coordination site. Furthermore, free energy calculations reproduced the experimentally derived monovalent cation selectivity. Our results suggest that Slo2.2 and Kir channels share a similar mechanism to coordinate Na(+). The localization of an Na(+) sensor within the RCK2 domain of Slo2.2 further supports the role of RCK (regulators of conductance of K(+)) domains of Slo channels in coupling ion sensing to channel gating.
    Journal of Neuroscience 06/2010; 30(22):7554-62. · 6.91 Impact Factor
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    ABSTRACT: The Slo3 gene encodes a high conductance potassium channel, which is activated by both voltage and intracellular alkalinization. Slo3 is specifically expressed in mammalian sperm cells, where it gives rise to pH-dependent outwardly rectifying K+ currents. Sperm Slo3 is the main current responsible for the capacitation-induced hyperpolarization, which is required for the ensuing acrosome reaction, an exocytotic process essential for fertilization. Here we show that in intact spermatozoa and in a heterologous expression system, the activation of Slo3 currents is regulated by PtdIns(4,5)P2 (PIP2). Depletion of endogenous PIP2 in inside-out macro-patches from Xenopus oocytes inhibited heterologously expressed Slo3 currents. Whole-cell recordings of sperm Slo3 currents or of Slo3 channels co-expressed in Xenopus oocytes with epidermal growth factor receptor (EGFR), demonstrated that stimulation by EGF could inhibit channel activity in a PIP2-dependent manner. High concentrations of PIP2 in the patch pipette not only resulted in a strong increase in sperm Slo3 current density, but also prevented the EGF-induced inhibition of this current. Mutation of positively charged residues involved in channel-PIP2 interactions enhanced the EGF-induced inhibition of Slo3 currents. Overall, our results suggest that PIP2 is an important regulator for Slo3 activation and that receptor-mediated hydrolysis of PIP2 leads to inhibition of Slo3 currents both in native and heterologous expression systems.
    Journal of Biological Chemistry 04/2010; · 4.65 Impact Factor
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    ABSTRACT: The Slo3 gene encodes a high conductance potassium channel, which is activated by both voltage and intracellular alkalinization. Slo3 is specifically expressed in mammalian sperm cells, where it gives rise to pH-dependent outwardly rectifying K(+) currents. Sperm Slo3 is the main current responsible for the capacitation-induced hyperpolarization, which is required for the ensuing acrosome reaction, an exocytotic process essential for fertilization. Here we show that in intact spermatozoa and in a heterologous expression system, the activation of Slo3 currents is regulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Depletion of endogenous PIP(2) in inside-out macropatches from Xenopus oocytes inhibited heterologously expressed Slo3 currents. Whole-cell recordings of sperm Slo3 currents or of Slo3 channels co-expressed in Xenopus oocytes with epidermal growth factor receptor, demonstrated that stimulation by epidermal growth factor (EGF) could inhibit channel activity in a PIP(2)-dependent manner. High concentrations of PIP(2) in the patch pipette not only resulted in a strong increase in sperm Slo3 current density but also prevented the EGF-induced inhibition of this current. Mutation of positively charged residues involved in channel-PIP(2) interactions enhanced the EGF-induced inhibition of Slo3 currents. Overall, our results suggest that PIP(2) is an important regulator for Slo3 activation and that receptor-mediated hydrolysis of PIP(2) leads to inhibition of Slo3 currents both in native and heterologous expression systems.
    Journal of Biological Chemistry 04/2010; 285(25):19259-66. · 4.65 Impact Factor
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    ABSTRACT: pH-regulated Slo3 channels, perhaps exclusively expressed in mammalian sperm, may play a role in alkalization-mediated K(+) fluxes associated with sperm capacitation. The Slo3 channel shares extensive homology with Ca(2+)- and voltage-regulated BK-type Slo1 K(+) channels. Here, using heterologous expression in oocytes, we define distinctive differences in pharmacological properties of Slo3 and Slo1 currents, examine blockade in terms of distinct blocking models, and, for some blockers, use mutated constructs to evaluate determinants of block. Slo3 is resistant to block by the standard Slo1 blockers, iberiotoxin, charybdotoxin and extracellular TEA. Slo3 is relatively insensitive to extracellular 4-AP up to 100 mM, while Slo1 is blocked in a voltage-dependent fashion consistent with block on the extracellular side of the channel. Block of both Slo1 and Slo3 by cytosolic 4-AP can be described by open channel block, with Slo3 being approximately 10-15-fold more sensitive, but exhibiting weaker voltage-dependence of block. The cytosolic concentrations of 4-AP required to block Slo3 make it unlikely that the effects of 4-AP on volume regulation in mammalian sperm is mediated by Slo3. Quinidine was more effective in blocking Slo3 than Slo1. For Slo1, quinidine block was favored by depolarization, irrespective of the side of application. For Slo3, quinidine block was relieved by depolarization, irrespective of the side of application, with strong block by less than 10 microM quinidine at potentials near 0 mV. The unusual voltage-dependence of block of Slo3 by quinidine may result from preferential binding of quinidine to closed Slo3 channels. The quinidine concentrations effective in blocking Slo3 suggest, that in experiments that have examined quinidine effects on sperm, any Slo3 currents would be almost completely inhibited.
    Channels (Austin, Tex.) 01/2010; 4(1):22-41. · 1.91 Impact Factor
  • Biophysical Journal 01/2010; 98(3). · 3.67 Impact Factor
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    ABSTRACT: N termini of auxiliary beta subunits that produce inactivation of large-conductance Ca(2+)-activated K(+) (BK) channels reach their pore-blocking position by first passing through side portals into an antechamber separating the BK pore module and the large C-terminal cytosolic domain. Previous work indicated that the beta2 subunit inactivation domain is protected from digestion by trypsin when bound in the inactivated conformation. Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber. Here, we provide additional tests of this model and examine its applicability to other beta subunit N termini. First, we show that specific mutations in the beta2 inactivation segment can speed up digestion by trypsin under closed-channel conditions, supporting the idea that the beta2 N terminus is protected by binding within the antechamber. Second, we show that cytosolic channel blockers distinguish between protection mediated by inactivation and protection under closed-channel conditions, implicating two distinct sites of protection. Together, these results confirm the idea that beta2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity. In contrast, the beta 3a N terminus is digested over 10-fold more quickly than the beta2 N terminus. Analysis of factors that contribute to differences in digestion rates suggests that binding of an N terminus within the antechamber constrains the trypsin accessibility of digestible basic residues, even when such residues are positioned outside the antechamber. Our analysis indicates that up to two N termini may simultaneously be protected from digestion. These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation.
    The Journal of General Physiology 04/2009; 133(3):263-82. · 4.73 Impact Factor
  • Biophysical Journal 01/2009; 96(3). · 3.67 Impact Factor
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    ABSTRACT: Inwardly rectifying potassium (Kir) channels are gated by the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). Among them, Kir3 requires additional molecules, such as the betagamma subunits of G proteins or intracellular sodium, for channel gating. Using an interactive computational-experimental approach, we show that sodium sensitivity of Kir channels involves the side chains of an aspartate and a histidine located across from each other in a crucial loop in the cytosolic domain, as well as the backbone carbonyls of two more residues and a water molecule. The location of the coordination site in the vicinity of a conserved arginine shown to affect channel-PtdIns(4,5)P(2) interactions suggests that sodium triggers a structural switch that frees the crucial arginine. Mutations of the aspartate and the histidine that affect sodium sensitivity also enhance the channel's sensitivity to PtdIns(4,5)P(2). Furthermore, on the basis of the molecular characteristics of the coordination site, we identify and confirm experimentally a sodium-sensitive phenotype in Kir5.1.
    Nature Chemical Biology 10/2008; 4(10):624-31. · 12.95 Impact Factor
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    ABSTRACT: Cytosolic N-terminal segments of many K+ channel subunits mediate rapid blockade of ion permeation by physical occlusion of the ion-conducting pore. For some channels with large cytosolic structures, access to the channel pore by inactivation domains may occur through lateral entry pathways or "side portals" that separate the pore domain and associated cytosolic structures covering the axis of the permeation pathway. However, the extent to which side portals control access of molecules to the channel or influence channel gating is unknown. Here we use removal of inactivation by trypsin as a tool to examine basic residue accessibility in both the N terminus of the native auxiliary beta2 subunit of Ca2+-activated, BK-type K+ channels and beta2 subunits with artificial inactivating N termini. The results show that, for BK channels, side portals define a protected space that precedes the channel permeation pathway and excludes small proteins such as trypsin but allows inactivation domains to enter. When channels are closed, inactivation domains readily pass through side portals, with a central antechamber preceding the permeation pathway occupied by an inactivation domain approximately half of the time under resting conditions. The restricted volume of the pathway through side portals is likely to influence kinetic properties of inactivation mechanisms, blockade by large pharmacological probes, and accessibility of modulatory factors to surfaces of the channel within the protected space.
    Journal of Neuroscience 12/2006; 26(46):11833-43. · 6.91 Impact Factor

Publication Stats

90 Citations
89.89 Total Impact Points

Institutions

  • 2010–2013
    • Virginia Commonwealth University
      • Department of Physiology and Biophysics
      Richmond, VA, United States
  • 2006–2010
    • Washington University in St. Louis
      • Department of Anesthesiology
      Saint Louis, MO, United States
  • 2008
    • Mount Sinai School of Medicine
      • Department of Structural and Chemical Biology
      Manhattan, NY, United States