Permeation properties of inward-rectifier potassium channels and their molecular determinants

The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K(+) channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K(+) conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single-channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH(2) terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl(+) > K(+) > Rb(+) > NH(4)(+). The ion selectivity sequence for ROMK2 based on conductance ratios was NH(4)(+) (1.6) > K(+) (1) > Tl(+) (0.5) > Rb(+) (0.4). For IRK1, the sequence was K(+) (1) > Tl(+) (0.8) > NH(4)(+) (0.6) > Rb(+) (0.1). The difference in the NH(4)(+)/ K(+) conductance (1.6) and permeability (0.09) ratios can be explained if NH(4)(+) binds with lower affinity than K(+) to sites within the pore. The relatively low conductances of NH(4)(+) and Rb(+) through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl(+) conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore.     

Molecular determinants of Rem2 regulation of N-type calcium channels

Rem2 belongs to the RGK family of small GTPases whose members are known to interact with the voltage gated calcium channel β subunit, and to inhibit or abolish calcium currents. To identify the underlying functional domains of Rem2, we created several N- or C-terminally truncated Rem2 proteins and examined their abilities to interact with the Ca_v β subunit and to regulate the activities of Ca_v2.2 N-type calcium channels. Confocal imaging of Rem2 in tsA-201 cells revealed that it contains a membrane-targeting signal in its C-terminus, consistent with previous studies. Co-precipitation assays showed that Ca_v β₃ interaction depends on Rem2 residues 1-123. Only Rem2 proteins that targeted the cell membrane as well as bound the β subunit were able to reduce whole cell calcium currents.     

Molecular determinants of syntaxin 1 modulation of N-type calcium channels

We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation (Jarvis, S. E., and Zamponi, G. W. (2001) J. Neurosci. 21, 2939-2948). Here, we have investigated the molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein beta subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to Gbeta and the N-terminal (residues 1-69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH(2)-terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both "open" and "closed" conformations during exocytosis, we produced a constitutively open form of syntaxin 1A and found that it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appeared to compete with Ggamma for the Gbeta subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein gamma subunit-like domain.     

Chemical determinants involved in anandamide-induced inhibition of T-type calcium channels

Anandamide, originally described as an endocannabinoid, is the main representative molecule of a new class of signaling lipids including endocannabinoids and N-acyl-related molecules, eicosanoids, and fatty acids. Bioactive lipids regulate neuronal excitability by acting on G-protein-coupled receptors (such as CB1) but also directly modulate various ionic conductances including voltage-activated T-type calcium channels (T-channels). However, little is known about the properties and the specificity of this new class of molecules on their various targets. In this study, we have investigated the chemical determinants involved in anandamide-induced inhibition of the three cloned T-channels: Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3. We show that both the hydroxyl group and the alkyl chain of anandamide are key determinants of its effects on T-currents. As follows, T-currents are also inhibited by fatty acids. Inhibition of the three Ca(V)3 currents by anandamide and arachidonic acid does not involve enzymatic metabolism and occurs in cell-free inside-out patches. Inhibition of T-currents by fatty acids and N-acyl ethanolamides depends on the degree of unsaturation but not on the alkyl chain length and consequently is not restricted to eicosanoids. Inhibition increases for polyunsaturated fatty acids comprising 18-22 carbons when cis-double bonds are close to the carboxyl group. Therefore the major natural (food-supplied) and mammalian endogenous fatty acids including gamma-linolenic acid, mead acid, and arachidonic acid as well as the fully polyunsaturated omega3-fatty acids that are enriched in fish oil eicosapentaenoic and docosahexaenoic acids are potent inhibitors of T-currents, which possibly contribute to their physiological functions.     

Voltage dependent calcium channels in cerebellar granule cell primary cultures

Voltage activated calcium channels were studied in rat cerebellar granule cells in primary culture. Macroscopic currents, carried by 20mM Ba2+, were measured in the whole-cell configuration. Slowly inactivating macroscopic currents, with a maximum value at a membrane potential around 5 mV, were recorded between the 1st and the 4th day in culture. These currents were completely blocked by 5mM Co2+, partially blocked by 10 microM nifedipine, and increased by 2 to 5 microM BAY K-8644. Two types of channels, in the presence of 80 mM Ba2+, were identified by single channel recording in cell-attached patches. The first type, which was dihydropyridine agonist sensitive, had a conductance of 18 pS, a half activation potential of more than 10 mV and did not inactivate. This type of channel was the only type found during the first four days in culture, although it was also present up to the 11th day. The second type of channel was dihydropyridine insensitive, had a conductance of 10 pS, a half activation potential less than -15 mV, and displayed voltage dependent inactivation. This second type of channel was found in cells for more than four days in culture.     

I-II loop structural determinants in the gating and surface expression of low voltage-activated calcium channels

The intracellular loops that interlink the four transmembrane domains of Ca(2+)- and Na(+)-channels (Ca(v), Na(v)) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I-II loop) in high voltage-activated (HVA) Ca(2+) channels possesses the binding site for Ca(v)beta subunits and plays significant roles in channel function, including trafficking the alpha(1) subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I-II loop of Ca(v)1/Ca(v)2 HVA channels and Ca(v)3 LVA/T-type channels, evidence for a regulatory role of the I-II loop in T-channel function has recently emerged for Ca(v)3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Ca(v)3 channels, we have performed a structure-function analysis of the I-II loop in Ca(v)3.1 and Ca(v)3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Ca(v)3.1 and Ca(v)3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Ca(v)3 channels. In contrast to findings in Ca(v)3.2, deletion of the central region of the I-II loop in Ca(v)3.1 and Ca(v)3.3 yielded a modest increase (+30%) and a reduction (-30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Ca(v)3 function by highlighting the unique role played by the intracellular I-II loop in Ca(v)3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Ca(v)3.3.     

Revealing the molecular determinants of neurotoxin specificity for calcium-activated versus voltage-dependent potassium channels

Potassium channel dysfunction underlies diseases such as epilepsy, hypertension, cardiac arrhythmias, and multiple sclerosis. Neurotoxins that selectively inhibit potassium channels, alpha-KTx, have provided invaluable information for dissecting the contribution of different potassium channels to neurotransmission, vasoconstriction, and lymphocyte proliferation. Thus, alpha-KTx specificity comprises an important first step in potassium channel-directed drug discovery for these diseases. Despite extensive functional and structural studies of alpha-KTx-potassium channel complexes, none have predicted the molecular basis of alpha-KTx specificity. Here we show that by minimizing the differences in binding free energy between selective and nonselective alpha-KTx we are able to identify all of the determinants of alpha-KTx specificity for calcium-activated versus voltage-dependent potassium channels. Because these determinants correspond to unique features of the two types of channels, they provide a way to develop more accurate models of alpha-KTx-potassium channel complexes that can be used to design novel selective alpha-KTx inhibitors.     

Subunits of purified calcium channels. Alpha 2 and delta are encoded by the same gene

Polyacrylamide gel electrophoresis of purified rabbit skeletal muscle L-type calcium channel before and after reduction of disulfide bonds confirmed that 27- and 24-kDa forms of the delta subunit are disulfide-linked to the 143-kDa alpha 2 subunit. The amino acid sequences of three peptides obtained by tryptic digestion of the delta subunits corresponded to amino acid sequences predicted from the 3' region of the mRNA encoding alpha 2. One of these peptides had the same sequence as the N terminus of the 24- and 27-kDa forms of the delta subunit and corresponded to residues 935-946 of the predicted alpha 2 primary sequence. Anti-peptide antibodies directed to regions on the N-terminal side of this site recognized the 143-kDa alpha 2 subunit in immunoblots of purified calcium channels under reducing conditions, whereas an antipeptide antibody directed toward a sequence on the C-terminal side of this site recognized 24- and 27-kDa forms of the delta subunit. A similar result was obtained after immunoblotting using purified transverse tubules or crude microsomal membrane preparations indicating that alpha 2 and delta occur as distinct disulfide-linked polypeptides in skeletal muscle membranes. Thus, the delta subunits are encoded by the same gene as the alpha 2 subunit and are integral components of the skeletal muscle calcium channel.     

Voltage activated calcium channels in somatic muscle of filarial nematode Setaria cervi

Acetylcholine (Ach), levamisole and pyrantel pamoate all cause stimulation of spontaneous rhythmic movements of whole worm and nerve muscle preparation of filarial nematode Setaria cervi. These stimulant effects are manifested only in the presence of available Ca2+ or extracellular Ca2+. Electrical stimulation of nerve muscle preparation of Setaria cervi elicited depolarization and increase in amplitude and tone of contractions. Electrical current stimulates Ca2+ entry leading to depolarization and during the phase of depolarization addition of any of the three stimulants viz. Ach, levamisole or pyrantel pamoate fails to elicit any response on nerve muscle preparation. The findings indicate that electrical stimulation, excitatory neurotransmitter Ach and stimulant anthelmintics levamisole and pyrantel pamoate all produce their stimulant effect by triggering entry of Ca2+ into the muscle cell. Further, blocking the calcium channels by nifedepine and thereby the entry of Ca2+ into the cells blocks the stimulant effect of Ach levamisole and pyrantel pamoate.     

Identification of inactivation determinants in the domain IIS6 region of high voltage-activated calcium channels

We have recently reported that transfer of the domain IIS6 region from rapidly inactivating R-type (alpha(1E)) calcium channels to slowly inactivating L-type (alpha(1C)) calcium channel confers rapid inactivation (Stotz, S. C., Hamid, J., Spaetgens, R. L., Jarvis, S. E., and Zamponi, G. W. (2000) J. Biol. Chem. 275, 24575-24582). Here we have identified individual amino acid residues in the IIS6 regions that are responsible for these effects. In this region, alpha(1C) and alpha(1E) channels differ in seven residues, and exchanging five of those residues individually or in combination did not significantly affect inactivation kinetics. By contrast, replacement of residues Phe-823 or Ile-829 of alpha(1C) with the corresponding alpha(1E) residues significantly accelerated inactivation rates and, when substituted concomitantly, approached the rapid inactivation kinetics of R-type channels. A systematic substitution of these residues with a series of other amino acids revealed that decreasing side chain size at position 823 accelerates inactivation, whereas a dependence of the inactivation kinetics on the degree of hydrophobicity could be observed at position 829. Although these point mutations facilitated rapid entry into the inactivated state of the channel, they had little to no effect on the rate of recovery from inactivation. This suggests that the development of and recovery from inactivation are governed by separate structural determinants. Finally, the effects of mutations that accelerated alpha(1C) inactivation could still be antagonized following coexpression of the rat beta(2a) subunit or by domain I-II linker substitutions that produce ultra slow inactivation of wild type channels, indicating that the inactivation kinetics seen with the mutants remain subject to regulation by the domain I-II linker. Overall, our results provide novel insights into a complex process underlying calcium channel inactivation.     

Ca(2+)-activated K+ channels: molecular determinants and function of the SK family

Ca(2+)-activated K(+) (K(Ca)) channels of small (SK) and intermediate (IK) conductance are present in a wide range of excitable and non-excitable cells. On activation by low concentrations of Ca(2+), they open, which results in hyperpolarization of the membrane potential and changes in cellular excitability. K(Ca)-channel activation also counteracts further increases in intracellular Ca(2+), thereby regulating the concentration of this ubiquitous intracellular messenger in space and time. K(Ca) channels have various functions, including the regulation of neuronal firing properties, blood flow and cell proliferation. The cloning of SK and IK channels has prompted investigations into their gating, pharmacology and organization into calcium-signalling domains, and has provided a framework that can be used to correlate molecularly identified K(Ca) channels with their native currents.     

A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels

Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 microM) of nickel were found to selectively block the Ca(v)3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Ca(v)3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Ca(v)3.2 and the nickel-insensitive Ca(v)3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Ca(v)3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3-S4 loop of domain I significantly attenuated the nickel block of Ca(v)3.2, mimicking the nickel-insensitive blocking potency of Ca(v)3.1. These findings indicate that His-191 in the S3-S4 loop is a critical residue conferring nickel block to Ca(v)3.2 and reveal a novel role for the S3-S4 loop to control ion permeation through T-type Ca2+ channels.     

Voltage control of Ca2+ permeation through N-type calcium (CaV2.2) channels

Voltage-gated calcium (Ca_V) channels deliver Ca²⁺ to trigger cellular functions ranging from cardiac muscle contraction to neurotransmitter release. The mechanism by which these channels select for Ca²⁺ over other cations is thought to involve multiple Ca²⁺-binding sites within the pore. Although the Ca²⁺ affinity and cation preference of these sites have been extensively investigated, the effect of voltage on these sites has not received the same attention. We used a neuronal preparation enriched for N-type calcium (Ca_V2.2) channels to investigate the effect of voltage on Ca²⁺ flux. We found that the EC₅₀ for Ca²⁺ permeation increases from 13 mM at 0 mV to 240 mM at 60 mV, indicating that, during permeation, Ca²⁺ ions sense the electric field. These data were nicely reproduced using a three-binding-site step model. Using roscovitine to slow Ca_V2.2 channel deactivation, we extended these measurements to voltages <0 mV. Permeation was minimally affected at these hyperpolarized voltages, as was predicted by the model. As an independent test of voltage effects on permeation, we examined the Ca²⁺-Ba²⁺ anomalous mole fraction (MF) effect, which was both concentration and voltage dependent. However, the Ca²⁺-Ba²⁺ anomalous MF data could not be reproduced unless we added a fourth site to our model. Thus, Ca²⁺ permeation through Ca_V2.2 channels may require at least four Ca²⁺-binding sites. Finally, our results suggest that the high affinity of Ca²⁺ for the channel helps to enhance Ca²⁺ influx at depolarized voltages relative to other ions (e.g., Ba²⁺ or Na⁺), whereas the absence of voltage effects at negative potentials prevents Ca²⁺ from becoming a channel blocker. Both effects are needed to maximize Ca²⁺ influx over the voltages spanned by action potentials.     

Voltage-gated calcium channels and disease

Voltage-gated calcium channels are a family of integral membrane calcium-selective proteins found in all excitable and many nonexcitable cells. Calcium influx affects membrane electrical properties by depolarizing cells and generally increasing excitability. Calcium entry further regulates multiple intracellular signaling pathways as well as the biochemical factors that mediate physiological functions such as neurotransmitter release and muscle contraction. Small changes in the biophysical properties or expression of calcium channels can result in pathophysiological changes leading to serious chronic disorders. In humans, mutations in calcium channel genes have been linked to a number of serious neurological, retinal, cardiac, and muscular disorders.     

Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca²⁺]_cyt) and [Ca²⁺]_cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G₁ phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca²⁺ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.     

Regulators of G protein signaling proteins as determinants of the rate of desensitization of presynaptic calcium channels.

Norepinephrine inhibits omega-conotoxin GVIA-sensitive presynaptic Ca2+ channels in chick dorsal root ganglion neurons through two pathways, one mediated by Go and the other by Gi. These pathways desensitize at different rates. We have found that recombinant Galpha interacting protein (GAIP) and regulators of G protein signaling (RGS)4 selectively accelerate the rate of desensitization of Go- and Gi-mediated pathways, respectively. Blockade of endogenous RGS proteins using antibodies raised against Galpha interacting protein and RGS4 slows the rate of desensitization of these pathways in a selective manner. These results demonstrate that different RGS proteins may interact with Gi and Go selectively, giving rise to distinct time courses of transmitter-mediated effects.     

Similar molecular determinants on Rem mediate two distinct modes of inhibition of CaV1.2 channels

Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like GTPases that potently inhibit all high-voltage-gated calcium (Ca_V1/Ca_V2) channels and are, thus, well-positioned to tune diverse physiological processes. Understanding how RGK proteins inhibit Ca_V channels is important for perspectives on their (patho)physiological roles and could advance their development and use as genetically-encoded Ca_V channel blockers. We previously reported that Rem can block surface Ca_V1.2 channels in 2 independent ways that engage distinct components of the channel complex: (1) by binding auxiliary β subunits (β-binding-dependent inhibition, or BBD); and (2) by binding the pore-forming α_1C subunit N-terminus (α_1C-binding-dependent inhibition, or ABD). By contrast, Gem uses only the BBD mechanism to block Ca_V1.2. Rem molecular determinants required for BBD Ca_V1.2 inhibition are the distal C-terminus and the guanine nucleotide binding G-domain which interact with the plasma membrane and Ca_Vβ, respectively. However, Rem determinants for ABD Ca_V1.2 inhibition are unknown. Here, combining fluorescence resonance energy transfer, electrophysiology, systematic truncations, and Rem/Gem chimeras we found that the same Rem distal C-terminus and G-domain also mediate ABD Ca_V1.2 inhibition, but with different interaction partners. Rem distal C-terminus interacts with α_1C N-terminus to anchor the G-domain which likely interacts with an as-yet-unidentified site. In contrast to some previous studies, neither the C-terminus of Rem nor Gem was sufficient to inhibit Ca_V1/Ca_V2 channels. The results reveal that similar molecular determinants on Rem are repurposed to initiate 2 independent mechanisms of Ca_V1.2 inhibition.