Alice Butler

TRPγ, a Drosophila TRP–Related Subunit, Forms a Regulated Cation Channel with TRPL

TRP and TRPL are two light-sensitive cation channel subunits required for the Drosophila photoresponse; however, our understanding of the identities, subunit composition, and function of the light-responsive channels is incomplete. To explain the residual photoresponse that remains in the trp mutant, a third TRP-related subunit has previously been proposed to function with TRPL. Here, we identify such a subunit, TRPgamma. We show that TRPgamma is highly enriched in photoreceptor cells and preferentially heteromultimerizes with TRPL in vitro and in vivo. The N-terminal domain of TRPgamma dominantly suppressed the TRPL-dependent photoresponse, indicating that TRPgamma-TRPL heteromultimers contribute to the photoresponse. While TRPL and TRPgamma homomultimers are constitutively active, we demonstrate that TRPL-TRPgamma heteromultimers form a regulated phospholipase C- (PLC-) stimulated channel.

The SLO3 sperm-specific potassium channel plays a vital role in male fertility

Here we show a unique example of male infertility conferred by a gene knockout of the sperm‐specific, pH‐dependent SLO3 potassium channel. In striking contrast to wild‐type sperm which undergo membrane hyperpolarization during capacitation, we found that SLO3 mutant sperm undergo membrane depolarization. Several defects in SLO3 mutant sperm are evident under capacitating conditions, including impaired motility, a bent “hairpin” shape, and failure to undergo the acrosome reaction (AR). The failure of AR is rescued by valinomycin which hyperpolarizes mutant sperm. Thus SLO3 is the principal potassium channel responsible for capacitation‐induced hyperpolarization, and membrane hyperpolarization is crucial to the AR.

SLO-2, a K+ channel with an unusual Cl- dependence.

The gating of different potassium channels depends on many diverse factors. We now report a unique example of a K+ channel with a Cl− dependence. The slo-2 gene was cloned from Caenorhabditis elegans and is widely expressed in both neurons and muscles; it was highly abundant, as suggested by its high representation in the C. elegans EST database. SLO-2, like its paralogue, SLO-1, was also dependent on Ca2+. We show by site-directed mutagenesis that its requirements for both Cl− and Ca2+ are synergistic and associated with the same functional domain. SLO-2s dependence on Cl− implies that intracellular Cl− homeostasis may be important in regulating cellular excitability through this unusual K+ channel.

Opposite Regulation of Slick and Slack K+ Channels by Neuromodulators

Slick (Slo2.1) and Slack (Slo2.2) are two novel members of the mammalian Slo potassium channel gene family that may contribute to the resting potentials of cells and control their basal level of excitability. Slo2 channels have sensors that couple channel activity to the intracellular concentrations of Na+ and Cl− ions (Yuan et al., 2003). We now report that activity of both Slo2 channels is controlled by neuromodulators through Gαq-protein coupled receptors (GqPCRs) (the M1 muscarinic receptor and the mGluR1 metabotropic glutamate receptor). Experiments coexpressing channels and receptors in Xenopus oocytes show that Slo2.1 and Slo2.2 channels are modulated in opposite ways: Slo2.1 is strongly inhibited, whereas Slo2.2 currents are strongly activated through GqPCR stimulation. Differential regulation involves protein kinase C (PKC); application of the PKC activator PMA, to cells expressing channels but not receptors, inhibits Slo2.1 whole-cell currents and increases Slo2.2 currents. Synthesis of a chimera showed that the distal carboxyl region of Slo2.1 controls the sensitivity of Slo2.1 to PMA. Slo2 channels have widespread expression in brain (Bhattacharjee et al., 2002, 2005). Using immunocytochemical techniques, we show coexpression of Slo2 channels with the GqPCRs in cortical and hippocampal brain sections and in cultured hippocampal neurons. The differential control of these novel channels by neurotransmitters may elicit long-lasting increases or decreases in neuronal excitability and, because of their widespread distribution, may provide a mechanism to activate or repress electrical activity in many systems of the brain.

Behavioral Defects in C. elegans egl-36 Mutants Result from Potassium Channels Shifted in Voltage-Dependence of Activation

Mutations in the C. elegans egl-36 gene result in defective excitation of egg-laying and enteric muscles. Dominant gain-of-function alleles inhibit enteric and egg-laying muscle contraction, whereas a putative null mutation has no observed phenotype. egl-36 encodes a Shaw-type (Kv3) voltage-dependent potassium channel subunit. In Xenopus oocytes, wild-type egl-36 expresses noninactivating channels with slow activation kinetics. One gain-of-function mutation causes a single amino acid substitution in S6, and the other causes a substitution in the cytoplasmic amino terminal domain. Both mutant alleles produce channels dramatically shifted in their midpoints of activation toward hyperpolarized voltages. An egl-36::gfp fusion is expressed in egg-laying muscles and in a pair of enteric muscle motor neurons. The mutant egl-36 phenotypes can thus be explained by expression in these cells of potassium channels that are inappropriately opened at hyperpolarized potentials, causing decreased excitability due to increased potassium conductance.

Genomic Organization of Nematode 4TM K+ Channelsa

ABSTRACT: As many as 50 genes in the C. elegans genome may encode K+ channels belonging to the novel structural class of two‐pore (4TM) channels. Many 4TM channels can be grouped into channel subfamilies. We analyzed 4TM channels in C. elegans using methods made possible by having complete genomic sequence. Two genes were chosen for comprehensive analysis, n2P16 and n2P17. By comparing the pattern of conservation in genomic DNA sequences between C. elegans and a closely related species, C. briggsae, we were able to identify all coding regions and predict the gene structure for these two genes. Given the extent of the 4TM channel family, we were surprised to discover that n2P17 produced at least six alternative transcripts encoding a constant central region and variable amino‐ and carboxyl‐termini. Blocks of highly conserved DNA sequences in noncoding regions were also apparent and most likely confer important regulatory functions. The interspecies comparison of the deduced channel proteins revealed that the extracellular loop between M1 and P1 is an apparent hot spot for evolutionary change in both channels. This contrasts with the membrane‐spanning domains that are highly conserved. Analysis of intron positions for 36 channels revealed that introns are frequently present at an identical position within the pore region, but very few are located in membrane‐spanning domains.

KCNQ-like Potassium Channels in Caenorhabditis elegans CONSERVED PROPERTIES AND MODULATION

The human KCNQ gene family encodes potassium channels linked to several genetic syndromes including neonatal epilepsy, cardiac arrhythmia, and progressive deafness. KCNQ channels form M-type potassium channels, which are critical regulators of neuronal excitability that mediate autonomic responses, pain, and higher brain function. Fundamental mechanisms of the normal and abnormal cellular roles for these channels may be gained from their study in simple model organisms. Here we report that a multigene family of KCNQ-like channels is present in the nematode, Caenorhabditis elegans. We show that many aspects of the functional properties, tissue expression pattern, and modulation of these C. elegans channels are conserved, including suppression by the M1 muscarinic receptor. We also describe a conserved mechanism of modulation by diacylglycerol for a subset of C. elegans and vertebrate KCNQ/KQT channels, which is dependent upon the carboxyl-terminal domains of channel subunits and activated protein kinase C.

SLO3 K+ Channels Control Calcium Entry through CATSPER Channels in Sperm

Background: SLO3 and CATSPER are two sperm-specific ion channels. Results: SLO3 K+ channels control Ca2+ entry through CATSPER channels. Conclusion: SLO3 control of CATSPER channel activity involves an intermediary step in which SLO3-dependent hyperpolarization may elicit internal alkalization via a voltage-dependent mechanism. Significance: Understanding the control of Ca2+ entry in sperm is crucial to understanding fertility; this study also reveals an unusual role for a K+ channel. Here we show how a sperm-specific potassium channel (SLO3) controls Ca2+ entry into sperm through a sperm-specific Ca2+ channel, CATSPER, in a totally unanticipated manner. The genetic deletion of either of those channels confers male infertility in mice. During sperm capacitation SLO3 hyperpolarizes the sperm, whereas CATSPER allows Ca2+ entry. These two channels may be functionally connected, but it had not been demonstrated that SLO3-dependent hyperpolarization is required for Ca2+ entry through CATSPER channels, nor has a functional mechanism linking the two channels been shown. In this study we show that Ca2+ entry through CATSPER channels is deficient in Slo3 mutant sperm lacking hyperpolarization; we also present evidence supporting the hypothesis that SLO3 channels activate CATSPER channels indirectly by promoting a rise in intracellular pH through a voltage-dependent mechanism. This mechanism may work through a Na+/H+ exchanger (sNHE) and/or a bicarbonate transporter, which utilizes the inward driving force of the Na+ gradient, rendering it intrinsically voltage-dependent. In addition, the sperm-specific Na+/H+ exchanger (sNHE) possess a putative voltage sensor that might be activated by membrane hyperpolarization, thus increasing the voltage sensitivity of internal alkalization.

Dissection of K+ currents in Caenorhabditis elegans muscle cells by genetics and RNA interference.

GFP-promoter experiments have previously shown that at least nine genes encoding potassium channel subunits are expressed in Caenorhabditis elegans muscle. By using genetic, RNA interference, and physiological techniques we revealed the molecular identity of the major components of the outward K+ currents in body wall muscle cells in culture. We found that under physiological conditions, outward current is dominated by the products of only two genes, Shaker (Kv1) and Shal (Kv4), both expressing voltage-dependent potassium channels. Other channels may be held in reserve to respond to particular circumstances. Because GFP-promoter experiments indicated that slo-2 expression is prominent, we created a deletion mutant to identify the SLO-2 current in vivo. In both whole-cell and single-channel modes, in vivo SLO-2 channels were active only when intracellular Ca2+ and Cl- were raised above normal physiological conditions, as occurs during hypoxia. Under such conditions, SLO-2 is the largest outward current, contributing up to 87% of the total current. Other channels are present in muscle, but our results suggest that they are unlikely to contribute a large outward component under physiological conditions. However, they, too, may contribute currents conditional on other factors. Hence, the picture that emerges is of a complex membrane with a small number of household conductances functioning under normal circumstances, but with additional conductances that are activated during unusual circumstances.

Properties of Slo1 K+ channels with and without the gating ring

Significance High-conductance Slo1 (BK) K+ channels are synergistically activated by Ca2+, voltage, Mg2+, and additional factors to modulate membrane excitability in many key physiological processes. Slo1 channels are of modular design with allosteric interactions between the core transmembrane modules and a large cytoplasmic gating ring module, providing a model system to study allosteric principles in channel gating and protein function. To examine the allosteric interactions, we developed constructs that replace the large gating ring module with short peptides and then characterized the altered properties of the gating. Our studies, which provide insight into functional and allosteric interactions between the core and the gating ring, may be useful in understanding the disease processes associated with Slo1-channel dysfunction. High-conductance Ca2+- and voltage-activated K+ (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca2+ and Mg2+ sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca2+- and Mg2+-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca2+ and Mg2+ activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.