Ji-Fang Zhang

Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons

This paper provides a brief overview of the diversity of voltage-gated Ca2+ channels and our recent work on neuronal Ca2+ channels with novel pharmacological and biophysical properties that distinguish them from L, N, P or T-type channels. The Ca2+ channel alpha 1 subunit known as alpha 1A or BI [Mori Y., Friedrich T., Kim M.-S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T., Mikoshiba K., Imoto K., Tanabe T. and Numa S. (1991) Nature 350, 398-402] is generally assumed to encode the P-type Ca2+ channel. However, we find that alpha 1A expressed in Xenopus oocytes differs from P-type channels in its kinetics of inactivation and its degree of sensitivity to block by the peptide toxins omega-Aga-IVA and omega-CTx-MVIIC [Sather W. A., Tanabe T., Zhang J.-F., Mori Y., Adams M. E. and Tsien R. W. (1993) Neuron 11, 291-303]. Thus, alpha 1A is capable of generating a Ca2+ channel with characteristics quite distinct from P-type channels. Doe-1, recently cloned from the forebrain of a marine ray, is another alpha 1 subunit which exemplifies a different branch of the Ca2+ channel family tree [Horne W. A., Ellinor P. T., Inman I., Zhou M., Tsien R. W. and Schwarz T. L. (1993) Proc. Natn. Acad. Sci. U.S.A. 90, 3787-3791]. When expressed in Xenopus oocytes, doe-1 forms a high voltage-activated (HVA) Ca2+ channel [Ellinor P. T., Zhang J.-F., Randall A. D., Zhou M., Schwarz T. L., Tsien R. W. and Horne W. (1993) Nature 363, 455-458]. It inactivates more rapidly than any previously expressed calcium channel and is not blocked by dihydropyridine antagonists or omega-Aga-IVA. Doe-1 current is reduced by omega-CTx-GVIA, but the inhibition is readily reversible and requires micromolar toxin, in contrast to this toxins potent and irreversible block of N-type channels. Doe-1 shows considerable sensitivity to block by Ni2+ or Cd2+. We have identified components of Ca2+ channel current in rat cerebellar granule neurons with kinetic and pharmacological features similar to alpha 1A and doe-1 in oocytes [Randall A. D., Wendland B., Schweizer F., Miljanich G., Adams M. E. and Tsien R. W. (1993) Soc. Neurosci. Abstr. 19, 1478]. The doe-1-like component (R-type current) inactivates much more quickly than L, N or P-type channels, and also differs significantly in its pharmacology.(ABSTRACT TRUNCATED AT 400 WORDS)

Distinctive biophysical and pharmacological properties of class A (BI) calcium channel α1 subunits

Transcripts for the class A Ca2+ channel alpha 1 subunit (also known as BI) are present at high levels in many parts of the mammalian CNS and are widely assumed to encode the P-type Ca2+ channel. To characterize the biophysical and pharmacological properties of alpha 1A channels, macroscopic and single-channel recordings were made in Xenopus oocytes injected with alpha 1A cRNA. alpha 1-specific properties were identified by making systematic comparisons with the more familiar class C alpha 1 subunit under the condition of a standard ancillary subunit (alpha 2/delta + beta) makeup. alpha 1A currents activate and inactivate more rapidly and display steeper voltage dependence of gating than alpha 1C currents. Unlike alpha 1C, alpha 1A channels are largely insensitive to dihydropyridines and FPL 64176, but respond to the cone snail peptide omega-CTx-MVIIC(SNX-230), a potent and fairly selective inhibitor. In comparison with P-type Ca2+ channels in rat cerebellar Purkinje cells, alpha 1A channels in oocytes are approximately 10(2)-fold less sensitive to omega-Aga-IVA and approximately 10-fold more sensitive to omega-CTx-MVIIC. alpha 1A channels are not inhibited by Bay K 8644 and inactivate much more rapidly than P-type Ca2+ channels. Thus, alpha 1A is capable of generating a Ca2+ channel phenotype quite different from P-type current.

Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions

Ca2+ channels display remarkable selectivity and permeability, traditionally attributed to multiple, discrete Ca2+ binding sites lining the pore. Each of the four pore-forming segments of Ca2+ channel alpha 1 subunits contains a glutamate residue that contributes to high-affinity Ca2+ interactions. Replacement of all four P-region glutamates with glutamine or alanine abolished micromolar Ca2+ block of monovalent current without revealing any additional independent high-affinity Ca2+ binding site. Pairwise replacements of the four glutamates excluded the hypothesis that they form two independent high-affinity sites. Systematic alterations of side-chain length, charge, and polarity by glutamate replacement with aspartate, glutamine, or alanine weakened the Ca2+ interaction, with considerable asymmetry from one repeat to another. The P-region glutamate in repeat I was unusual in its sensitivity to aspartate replacement but not glutamine substitution. While all four glutamates cooperate in supporting high-affinity interactions with single Ca2+ ions, they also influence the interaction between multiple divalent cations.

Multiple Structural Elements in Voltage-Dependent Ca2+ Channels Support Their Inhibition by G Proteins

Molecular determinants of Ca2+ channel responsiveness to inhibition by receptor-coupled G proteins were investigated in Xenopus oocytes. The inhibitory response of alpha1B (N-type) channels was much larger than alpha1A (P/Q-type) channels, while alpha1C (L-type) channels were unresponsive. Differences in both degree and speed of inhibition were accounted for by variations in inhibitor off-rate. We tested proposals that inhibitory G protein and Ca2+ channel beta subunits compete specifically at the I-II loop. G protein-mediated inhibition remained unaltered in alpha1B subunits containing a point mutation in the I-II loop segment critical for Ca2+ channel beta subunit binding, and in chimeras where the I-II loop of alpha1B was replaced with counterparts from alpha1A or alpha1c. Full interconversion between modulatory behaviors of alpha1B and alpha1A was achieved only by swapping both motif I and the C-terminus in combination. Thus, essential structural elements for G protein modulation reside in multiple Ca2+ channel domains.

Modulation of the Cardiac Sodium Channel NaV1.5 by Fyn, a Src Family Tyrosine Kinase

Dynamic modulation of ion channels can produce dramatic alterations of electrical excitability in cardiac myocytes. This study addresses the effects of the Src family tyrosine kinase Fyn on NaV1.5 cardiac sodium channels. Sodium currents were acquired by whole cell recording on HEK-293 cells transiently expressing NaV1.5. Acute treatment of cells with insulin caused a depolarizing shift in steady-state inactivation, an effect eliminated by the Src-specific tyrosine kinase inhibitor PP2. Sodium channels were coexpressed with either constitutively active (FynCA) or catalytically inactive (FynKD) variants of Fyn. FynCA caused a 10-mV depolarizing shift of steady-state inactivation compared with FynKD without altering the activation conductance-voltage relationship. Comparable effects of these Fyn variants were obtained with whole-cell and perforated-patch recording. Tyrosine phosphorylation of immunoprecipitated NaV1.5 was increased in cells expressing FynCA compared with FynKD. We show that Fyn is present in rat cardiac myocytes, and that NaV1.5 channels from these myocytes are tyrosine-phosphorylated. In HEK-293 cells the effect of FynCA on NaV1.5 inactivation is abolished by the single point mutation Y1495F, a residue located within the cytoplasmic linker between the third and fourth homologous domains of the sodium channel. We provide evidence that this linker is a substrate for Fyn in vitro, and that Y1495 is a preferred phosphorylation site. These results suggest that cardiac sodium channels are physiologically relevant targets of Src family tyrosine kinases.

Structural Basis for Calmodulin as a Dynamic Calcium Sensor

Calmodulin is a prototypical and versatile Ca(2+) sensor with EF hands as its high-affinity Ca(2+) binding domains. Calmodulin is present in all eukaryotic cells, mediating Ca(2+)-dependent signaling. Upon binding Ca(2+), calmodulin changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on calmodulin, little is known on how target proteins regulate calmodulins ability to bind Ca(2+). Here, we take advantage of two splice variants of SK2 channels, which are activated by Ca(2+)-bound calmodulin but show different sensitivity to Ca(2+) for their activation. Protein crystal structures and other experiments show that, depending on which SK2 splice variant it binds to, calmodulin adopts drastically different conformations with different affinities for Ca(2+) at its C-lobe. Such target protein-induced conformational changes make calmodulin a dynamic Ca(2+) sensor capable of responding to different Ca(2+) concentrations in cellular Ca(2+) signaling.

Identification of the Functional Binding Pocket for Compounds Targeting Small-Conductance Ca2+-Activated Potassium Channels

Small- and intermediate-conductance Ca2+-activated potassium channels, activated by Ca2+-bound calmodulin, play an important role in regulating membrane excitability. These channels are also linked to clinical abnormalities. A tremendous amount of effort has been devoted to developing small molecule compounds targeting these channels. However, these compounds often suffer from low potency and lack of selectivity, hindering their potentials for clinical use. A key contributing factor is the lack of knowledge of the binding site(s) for these compounds. Here we demonstrate by X-ray crystallography that the binding pocket for the compounds of the 1-EBIO class is located at the calmodulin-channel interface. We show that, based on structure data and molecular docking, mutations of the channel can effectively change the potency of these compounds. Our results provide insight into the molecular nature of the binding pocket and its contribution to the potency and selectivity of the compounds of the 1-EBIO class.

Unstructured to Structured Transition of an Intrinsically Disordered Protein Peptide in Coupling Ca2+-sensing and SK Channel Activation

Most proteins, such as ion channels, form well-organized 3D structures to carry out their specific functions. A typical voltage-gated potassium channel subunit has six transmembrane segments (S1–S6) to form the voltage-sensing domain and the pore domain. Conformational changes of these domains result in opening of the channel pore. Intrinsically disordered (ID) proteins/peptides are considered equally important for the protein functions. However, it is difficult to explore the structural features underlying the functions of ID proteins/peptides by conventional methods, such as X-ray crystallography, because of the flexibility of their secondary structures. Unlike voltage-gated potassium channels, families of small- and intermediate-conductance Ca2+-activated potassium (SK/IK) channels with important roles in regulating membrane excitability are activated exclusively by Ca2+-bound calmodulin (CaM). Upon binding of Ca2+ to CaM, a 2 × 2 structure forms between CaM and the CaM-binding domain. A channel fragment that connects S6 and the CaM-binding domain is not visible in the protein crystal structure, suggesting that this fragment is an ID fragment. Here we show that the conformation of the ID fragment in SK channels becomes readily identifiable in the presence of NS309, the most potent compound that potentiates the channel activities. This well-defined conformation of the ID fragment, stabilized by NS309, increases the channel open probability at a given Ca2+ concentration. Our results demonstrate that the ID fragment, itself a target for drugs modulating SK channel activities, plays a unique role in coupling Ca2+ sensing by CaM and mechanical opening of SK channels.

Selective Phosphorylation Modulates the PIP2 Sensitivity of the CaM-SK Channel Complex

Phosphatidylinositol bisphosphate (PIP2) regulates the activities of many membrane proteins, including ion channels, through direct interactions. However, the affinity of PIP2 is so high for some channel proteins that its physiological role as a modulator has been questioned. Here we show that PIP2 is a key cofactor for activation of small conductance Ca2+-activated potassium channels (SKs) by Ca(2+)-bound calmodulin (CaM). Removal of the endogenous PIP2 inhibits SKs. The PIP2-binding site resides at the interface of CaM and the SK C terminus. We further demonstrate that the affinity of PIP2 for its target proteins can be regulated by cellular signaling. Phosphorylation of CaM T79, located adjacent to the PIP2-binding site, by casein kinase 2 reduces the affinity of PIP2 for the CaM-SK channel complex by altering the dynamic interactions among amino acid residues surrounding the PIP2-binding site. This effect of CaM phosphorylation promotes greater channel inhibition by G protein-mediated hydrolysis of PIP2.

Characterization of two distinct modes of endophilin in clathrin-mediated endocytosis

Endophilin, one of the main accessory proteins involved in clathrin-mediated endocytosis, interacts with other endocytic proteins, such as dynamin, by its SH3 domain. We previously reported that voltage-gated Ca(2+) channels are an integral part of the synaptic vesicle (SV) endocytosis machinery through their interaction with endophilin. Formation of the endophilin-channel complex is Ca(2+) dependent. A glutamate residue, E264, in endophilin is part of the primary Ca(2+) sensor for Ca(2+)-dependent formation of the channel-endophilin complex. We proposed that endophilin exists in two distinct modes (conformations), an open mode in the absence of Ca(2+), and a closed mode in the presence of Ca(2+). Binding of Ca(2+) switches endophilin from its open mode to the closed mode, resulting in dissociation of endophilin from other proteins. The present study is aimed at understanding the functional roles of endophilin in its two different modes, by creating two endophilin mutants, E264A and E264R, to mimic endophilin in its permanent open mode and permanent closed mode respectively. Here, we show that these two modes of endophilin have different effects on how endophilin interacts with other proteins, such as dynamin or β1-adrenergic receptors. In living cells, endophilin in its permanent closed mode does not show obvious effects on agonist-induced internalization of β1-adrenergic receptors. Endophilin, when in its permanent open mode, enhances the short-term synaptic depression in cultured hippocampal neurons, due partly to its failure to dissociate from Ca(2+) channels in the presence of Ca(2+). Our results show that modal switching by Ca(2+) allows endophilin to regulate, more effectively, the clathrin-mediated endocytosis of SV at the nerve terminal.