Luba Krapivinsky

TRPC1 and TRPC5 Form a Novel Cation Channel in Mammalian Brain

TRP proteins are cation channels responding to receptor-dependent activation of phospholipase C. Mammalian (TRPC) channels can form hetero-oligomeric channels in vitro, but native TRPC channel complexes have not been identified to date. We demonstrate here that TRPC1 and TRPC5 are subunits of a heteromeric neuronal channel. Both TRPC proteins have overlapping distributions in the hippocampus. Coexpression of TRPC1 and TRPC5 in HEK293 cells resulted in a novel nonselective cation channel with a voltage dependence similar to NMDA receptor channels, but unlike that of any reported TRPC channel. TRPC1/TRPC5 heteromers were activated by G(q)-coupled receptors but not by depletion of intracellular Ca(2+) stores. In contrast to the more common view of the TRP family as comprising store-operated channels, we propose that many TRPC heteromers form diverse receptor-regulated nonselective cation channels in the mammalian brain.

The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1.

The NMDA subtype of glutamate receptors (NMDAR) at excitatory neuronal synapses plays a key role in synaptic plasticity. The extracellular signal-regulated kinase (ERK1,2 or ERK) pathway is an essential component of NMDAR signal transduction controlling the neuroplasticity underlying memory processes, neuronal development, and refinement of synaptic connections. Here we show that NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts in vivo and in vitro with RasGRF1, a Ca(2+)/calmodulin-dependent Ras-guanine-nucleotide-releasing factor. Specific disruption of this interaction in living neurons abrogates NMDAR-dependent ERK activation. Thus, RasGRF1 serves as NMDAR-dependent regulator of the ERK kinase pathway. The specific association of RasGRF1 with the NR2B subunit and study of ERK activation in neurons with varied content of NR2B suggests that NR2B-containing channels are the dominant activators of the NMDA-dependent ERK pathway.

Formation of Novel TRPC Channels by Complex Subunit Interactions in Embryonic Brain

Mammalian short TRP channels (TRPCs) are putative receptor- and store-operated cation channels that play a fundamental role in the regulation of cellular Ca2+ homeostasis. Assembly of the seven TRPC homologs (TRPC1-7) into homo- and heteromers can create a large variety of different channels. However, the compositions as well as the functional properties of native TRPC complexes are largely undefined. We performed a systematic biochemical study of TRPC interactions in mammalian brain and identified previously unrecognized channel heteromers composed of TRPC1, TRPC4, or TRPC5 and the diacylglycerol-activated TRPC3 or TRPC6 subunits. The novel TRPC heteromers were found exclusively in embryonic brain. In heterologous systems, we demonstrated that assembly of these novel heteromers required the combination of TRPC1 plus TRPC4 or TRPC5 subunits along with diacylglycerol-sensitive subunits in the channel complexes. Functional interaction of the TRPC subunits was verified using a dominant negative TRPC5 mutant (TRPC5DN). Co-expression of TRPC5DN suppressed currents through TRPC5- and TRPC4-containing complexes; TRPC3-associated currents were unaffected by TRPC5DN unless TRPC1 was also co-expressed. This complex assembly mechanism increases the diversity of TRPC channels in mammalian brain and may generate novel heteromers that have specific roles in the developing brain.

All four CatSper ion channel proteins are required for male fertility and sperm cell hyperactivated motility

Mammalian spermatozoa become motile at ejaculation, but before they can fertilize the egg, they must acquire more thrust to penetrate the cumulus and zona pellucida. The forceful asymmetric motion of hyperactivated spermatozoa requires Ca2+ entry into the sperm tail by an alkalinization-activated voltage-sensitive Ca2+-selective current (ICatSper). Hyperactivation requires CatSper1 and CatSper2 putative ion channel genes, but the function of two other related genes (CatSper3 and CatSper4) is not known. Here we show that targeted disruption of murine CatSper3 or CatSper4 also abrogated ICatSper, sperm cell hyperactivated motility and male fertility but did not affect spermatogenesis or initial motility. Direct protein interactions among CatSpers, the sperm specificity of these proteins, and loss of ICatSper in each of the four CatSper−/− mice indicate that CatSpers are highly specialized flagellar proteins.

SynGAP-MUPP1-CaMKII Synaptic Complexes Regulate p38 MAP Kinase Activity and NMDA Receptor- Dependent Synaptic AMPA Receptor Potentiation

The synapse contains densely localized and interacting proteins that enable it to adapt to changing inputs. We describe a Ca2+-sensitive protein complex involved in the regulation of AMPA receptor synaptic plasticity. The complex is comprised of MUPPI, a multi-PDZ domain-containing protein; SynGAP, a synaptic GTPase-activating protein; and the Ca2+/calmodulin-dependent kinase CaMKII. In synapses of hippocampal neurons, SynGAP and CaMKII are brought together by direct physical interaction with the PDZ domains of MUPP1, and in this complex, SynGAP is phosphorylated. Ca2+CaM binding to CaMKII dissociates it from the MUPP1 complex, and Ca2+ entering via the NMDAR drives the dephosphorylation of SynGAP. Specific peptide-induced SynGAP dissociation from the MUPP1-CaMKII complex results in SynGAP dephosphorylation accompanied by P38 MAPK inactivation, potentiation of synaptic AMPA responses, and an increase in the number of AMPAR-containing clusters in hippocampal neuron synapses. siRNA-mediated SynGAP knockdown confirmed these results. These data implicate SynGAP in NMDAR- and CaMKII-dependent regulation of AMPAR trafficking.

The TRPM7 Ion Channel Functions in Cholinergic Synaptic Vesicles and Affects Transmitter Release

A longstanding hypothesis is that ion channels are present in the membranes of synaptic vesicles and might affect neurotransmitter release. Here we demonstrate that TRPM7, a member of the transient receptor potential (TRP) ion channel family, resides in the membrane of synaptic vesicles of sympathetic neurons, forms molecular complexes with the synaptic vesicle proteins synapsin I and synaptotagmin I, and directly interacts with synaptic vesicular snapin. In sympathetic neurons, changes in TRPM7 levels and channel activity alter acetylcholine release, as measured by EPSP amplitudes and decay times in postsynaptic neurons. TRPM7 affects EPSP quantal size, an intrinsic property of synaptic vesicle release. Targeted peptide interference of TRPM7s interaction with snapin affects the amplitudes and kinetics of postsynaptic EPSPs. Thus, vesicular TRPM7 channel activity is critical to neurotransmitter release in sympathetic neurons.

A novel inward rectifier K^+ channel with unique pore properties

We have cloned a novel K+-selective, inward rectifier channel that is widely expressed in brain but is especially abundant in the Purkinje cell layer of the cerebellum and pyramidal cells of the hippocampus. It is also present in a wide array of tissues, including kidney and intestine. The channel is only 38% identical to its closest relative, Kir1.3 (Kir1-ATP-regulated inward rectifier K+ [ROMK] family) and displays none of the functional properties unique to the ROMK class. Kir7.1 has several unique features, including a very low estimated single channel conductance (approximately 50 fS), low sensitivity to block by external Ba2+ and Cs+, and no dependence of its inward rectification properties on the internal blocking particle Mg2+. The unusual pore properties of Kir7.1 seem to be explained by amino acids in the pore sequence that differ from corresponding conserved residues in all other Kir channel proteins. Replacement of one of these amino acids (Met-125) with the Arg absolutely conserved in all other Kir channels dramatically increases its single channel conductance and Ba2+ sensitivity. This channel would provide a steady background K+ current to help set the membrane potential in cells in which it is expressed. We propose that the novel channel be assigned to a new Kir subfamily, Kir7.1.

A novel gene required for male fertility and functional CATSPER channel formation in spermatozoa.

Summary Calcium signaling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility, and the acrosome reaction are all mediated by increases in intracellular Ca2+. Cation channels of sperm proteins (CATSPERS1-4) form an alkalinization-activated Ca2+-selective channel required for the hyperactivated motility of spermatozoa and male fertility. Each of the CatSper1-4 genes encodes a subunit of a tetramer surrounding a Ca2+-selective pore, in analogy with other six-transmembrane ion channel α subunits. In addition to the pore-forming proteins, the sperm Ca2+ channel contains auxiliary subunits, CATSPERβ and CATSPERγ. Here, we identify the Tmem146 gene product as a novel subunit, CATSPERδ, required for CATSPER channel function. We find that mice lacking the sperm tail-specific CATSPERδ are infertile and their spermatozoa lack both Ca2+ current and hyperactivated motility. We show that CATSPERδ is an essential element of the CATSPER channel complex and propose that CATSPERδ is required for proper CATSPER channel assembly and/or transport.

pICln Inhibits snRNP Biogenesis by Binding Core Spliceosomal Proteins

ABSTRACT The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) form essential components of spliceosomes, the machinery that removes introns from pre-mRNAs in eukaryotic cells. A critical initial step in the complex process of snRNP biogenesis is the assembly of a group of common core proteins (Sm proteins) on spliceosomal snRNA. In this study we show by multiple independent methods that the protein pICln associates with Sm proteins in vivo and in vitro. The binding of pICln to Sm proteins interferes with Sm protein assembly on spliceosomal snRNAs and inhibits import of snRNAs into the nucleus. Furthermore, pICln prevents the interaction of Sm proteins with the survival of motor neurons (SMN) protein, an interaction that has been shown to be critical for snRNP biogenesis. These findings lead us to propose a model in which pICln participates in the regulation of snRNP biogenesis, at least in part by interfering with Sm protein interaction with SMN protein.

Cleavage of TRPM7 releases the kinase domain from the ion channel and regulates its participation in Fas-induced apoptosis

Transient receptor potential melastatin-like 7 (TRPM7) is a channel protein that also contains a regulatory serine-threonine kinase domain. Here, we find that Trpm7-/- T cells are deficient in Fas-receptor-induced apoptosis and that TRPM7 channel activity participates in the apoptotic process and is regulated by caspase-dependent cleavage. This function of TRPM7 is dependent on its function as a channel, but not as a kinase. TRPM7 is cleaved by caspases at D1510, disassociating the carboxy-terminal kinase domain from the pore without disrupting the phosphotransferase activity of the released kinase but substantially increasing TRPM7 ion channel activity. Furthermore, we show that TRPM7 regulates endocytic compartmentalization of the Fas receptor after receptor stimulation, an important process for apoptotic signaling through Fas receptors. These findings raise the possibility that other members of the TRP channel superfamily are also regulated by caspase-mediated cleavage, with wide-ranging implications for cell death and differentiation.