Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous cation channels that mediate release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and thereby are involved in many physiological processes. In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP3R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP3 sensitivity. We hypothesized the IP3R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP3R1 regulation by IP3, cytosolic, and luminal Ca2+ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca2+ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP3 ligand sensitivity. Single-channel IP3R1 studies revealed that the conductance of IP3R1-WT and -D2594K channels is similar. However, IP3R1-D2594K channels exhibit higher IP3 sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP3R1-D2594K showed a bell-shape cytosolic Ca2+-dependency, but D2594K had greater activity at each tested cytosolic free Ca2+ concentration. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels\' pore cytosolic exit affects the channel\'s gating behavior thereby explaining the enhanced ligand-channel\'s sensitivity.

The human transient receptor potential canonical 5 (TRPC5) is a calcium-permeable, nonselective cation channel expressed in the central and peripheral nervous system and also in other tissues such as the kidney, synovium, and odontoblasts. TRPC5 has been recently confirmed to play a key role in spontaneous, inflammatory mechanical, and cold pain. Although TRPC5 activation is known to be cold sensitive, it is unclear whether this property is intrinsic to the channel protein and whether or to what extent it may be determined by the cellular environment. In this study, we explored the cold sensitivity of human TRPC5 at the single-channel level using transiently transfected HEK293T cells. Upon decreasing the temperature, the channel demonstrated prolonged mean open dwell times and a robust increase in the open probability (Po ), whereas the amplitude of unitary currents decreased ~1.5-fold per 10°C of temperature difference. In the absence of any agonists, the temperature dependence of Po was sigmoidal, with a steep slope within the temperature range of 16°C-11°C, and exhibited saturation below 8-5°C. Thermodynamic analysis revealed significant changes in enthalpy and entropy, suggesting that substantial conformational changes accompany cold-induced gating. The mutant channel T970A, in which the regulation downstream of G-protein coupled receptor signaling was abrogated, exhibited higher basal activity at room temperature and a less steep temperature response profile, with an apparent threshold below 22°C. An even more pronounced decrease in the activation threshold was observed in a mutant that disrupted the electrostatic interaction of TRPC5 with the endoplasmic reticulum calcium sensor stromal interaction molecule 1. Thus, TRPC5 exhibits features of an intrinsically cold-gated channel; its sensitivity to cold tightly depends on the phosphorylation status of the protein and intracellular calcium homeostasis.

Potassium channels play an important role regulating transmembrane electrical activity in essentially all cell types. We were especially interested in those that determine the intrinsic electrical properties of mammalian central neurons. Over 30 different potassium channels have been molecularly identified in brain neurons, but there often is not a clear distinction between molecular structure and the function of a particular channel in the cell. Using patch-clamp methods to search for single potassium channels in excised inside-out (ISO) somatic patches with symmetrical potassium, we found that nearly all patches contained non-voltage-inactivating channels with a single-channel conductance of 100-200 pS. This conductance range is consistent with the family of sodium-activated potassium channels (Slo2.1, Slo2.2, or collectively, KNa). The activity of these channels was positively correlated with a low cytoplasmic Na+ concentration (2-20 mM). Cell-attached recordings from intact neurons, however, showed little or no activity of this K+ channel. Attempts to increase channel activity by increasing intracellular sodium concentration ([Na+]i) with bursts of action potentials or direct perfusion of Na+ through a whole cell pipette had little effect on KNa channel activity. Furthermore, excised outside-out (OSO) patches across a range of intracellular [Na+] showed less channel activity than we had seen with excised ISO patches. Blocking the Na+/K+ pump with ouabain increased the activity of the KNa channels in excised OSO patches to levels comparable with ISO-excised patches. Our results suggest that despite their apparent high levels of expression, the activity of somatic KNa channels is tightly regulated by the activity of the Na+/K+ pump.NEW & NOTEWORTHY We studied KNa channels in mouse hippocampal CA1 neurons. Excised inside-out patches showed the channels to be prevalent and active in most patches in the presence of Na+. Cell-attached recordings from intact neurons, however, showed little channel activity. Increasing cytoplasmic sodium in intact cells showed a small effect on channel activity compared with that seen in inside-out excised patches. Blockade of the Na+/K+ pump with ouabain, however, restored the activity of the channels to that seen in inside-out patches.

The IKs channel complex is formed by the co-assembly of Kv7.1 (KCNQ1), a voltage-gated potassium channel, with its β-subunit, KCNE1 and the association of numerous accessory regulatory molecules such as PIP2, calmodulin, and yotiao. As a result, the IKs potassium current shows kinetic and regulatory flexibility, which not only allows IKs to fulfill physiological roles as disparate as cardiac repolarization and the maintenance of endolymph K+ homeostasis, but also to cause significant disease when it malfunctions. Here, we review new areas of understanding in the assembly, kinetics of activation and inactivation, voltage-sensor pore coupling, unitary events and regulation of this important ion channel complex, all of which have been given further impetus by the recent solution of cryo-EM structural representations of KCNQ1 alone and KCNQ1+KCNE3. Recently, the stoichiometric ratio of KCNE1 to KCNQ1 subunits has been confirmed to be variable up to a ratio of 4:4, rather than fixed at 2:4, and we will review the results and new methodologies that support this conclusion. Significant advances have been made in understanding differences between KCNQ1 and IKs gating using voltage clamp fluorimetry and mutational analysis to illuminate voltage sensor activation and inactivation, and the relationship between voltage sensor translation and pore domain opening. We now understand that the KCNQ1 pore can open with different permeabilities and conductance when the voltage sensor is in partially or fully activated positions, and the ability to make robust single channel recordings from IKs channels has also revealed the complicated pore subconductance architecture during these opening steps, during inactivation, and regulation by 1-4 associated KCNE1 subunits. Experiments placing mutations into individual voltage sensors to drastically change voltage dependence or prevent their movement altogether have demonstrated that the activation of KCNQ1 alone and IKs can best be explained using allosteric models of channel gating. Finally, we discuss how the intrinsic gating properties of KCNQ1 and IKs are highly modulated through the impact of intracellular signaling molecules and co-factors such as PIP2, protein kinase A, calmodulin and ATP, all of which modulate IKs current kinetics and contribute to diverse IKs channel complex function.

TRPC5 channels are nonselective cation channels activated by G-protein-coupled receptors. It was previously found that recombinant TRPC5 currents are inhibited by intracellular ATP, when studied by whole-cell patch-clamp recording. In the present study, we investigated the mechanism of ATP inhibition at the single-channel level using patches from HEK-293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. In inside-out patches, application of ATP to the intracellular face of the membrane reduced TRPC5 channel activity at both positive and negative potentials without affecting the unitary current amplitude or open dwell time of the channel. The effect of ATP was rapidly reversible. These results suggest that ATP may bind to the channel protein and affect the ability of the channel to open or to remain in an open, nondesensitized state. The activity of TRPC5 channels may be influenced by cellular metabolism via changes in ATP levels.

Controlling the molecular interactions through protein nanopores is crucial for effectively detecting single molecules. Here, the development of a hetero-oligomeric nanopore derived from Nocardia farcinica porin AB (NfpAB) is discussed for single-molecule sensing of biopolymers. Using single-channel recording, the interaction of cyclic oligosaccharides such as cationic cyclodextrins (CDs) of different symmetries and charges with NfpAB is measured. Studies of the transport kinetics of CDs reveal asymmetric geometry and charge distribution of NfpAB. The applied potential promotes the attachment of the cationic CDs to the negatively charged pore surface due to electrostatic interaction. Further, the attached CDs are released from the pore by reversing the applied potential in time-resolved blockages. Release of CDs from the pore depends on its charge, size, and magnitude of the applied potential. The kinetics of CD attachment and release is controlled by fine-tuning the applied potential demonstrating the successful molecular transport across these nanopores. It is suggested that such controlled molecular interactions with protein nanopores using organic templates can be useful for several applications in nanopore technology and single-molecule chemistry.

Store-operated channels activated in response to intracellular calcium store depletion represent the main pathway of calcium entry from the extracellular space in nonelectroexcitable cells. Adapter proteins organize the components of this system into integral complex. We studied the influence of adapter proteins of the Homer family on endogenous store-operated calcium Imin channels in A431 cells. Monomeric Homer 1a proteins increase activity of Imin channels, but did not modulate their electrophysiological properties. Recombinant Homer 1c protein did not block the induced calcium currents.

Throughout postnatal maturation of the mouse inner ear, cochlear hair cells display at least two types of mechanically gated ion channel: normal mechanotransducer (MT) channels at the tips of the stereocilia, activated by tension in interciliary tip links, and anomalous mechanosensitive (MS) channels on the top surface of the cells. The anomalous MS channels are responsible for the reverse-polarity current that appears in mutants in which normal transduction is lost. They are also seen in wild-type hair cells around birth, appearing 2 days earlier than normal MT channels, and being down-regulated with the emergence of the normal channels. We review the evidence that the normal and anomalous channels are distinct channel types, which includes differences in localization, susceptibility to pharmacological agents, single-channel conductance and Ca2+ permeability. The dichotomy is reinforced by the observation that the anomalous current is absent in cochlear cells of Piezo2-null mice, even though the normal MT current persists. The anomalous current is suppressed by high intracellular Ca2+ , suggesting that influx of the divalent ion via more Ca2+ -permeable normal MT channels inhibits the anomalous channels, thus explaining the temporal relationship between the two. Piezo2-null mice have largely normal hearing, exhibiting up to 20 dB elevation in threshold in the acoustic brainstem response, so raising questions about the significance of PIEZO2 in the cochlea. Since the anomalous current declines with postnatal age, PIEZO2 may contribute to hair cell development, but it does not underlie the normal MT current. Its role in the development of hearing is not understood.

AMPA-type glutamate receptors are ligand-gated cation channels responsible for a majority of the fast excitatory synaptic transmission in the brain. Their behavior and calcium permeability depends critically on their subunit composition and the identity of associated auxiliary proteins. Calcium-permeable AMPA receptors (CP-AMPARs) contribute to various forms of synaptic plasticity, and their dysfunction underlies a number of serious neurological conditions. For CP-AMPARs, the prototypical transmembrane AMPAR regulatory protein stargazin, which acts as an auxiliary subunit, enhances receptor function by increasing single-channel conductance, slowing channel gating, increasing calcium permeability, and relieving the voltage-dependent block by endogenous intracellular polyamines. We find that, in contrast, GSG1L, a transmembrane auxiliary protein identified recently as being part of the AMPAR proteome, acts to reduce the weighted mean single-channel conductance and calcium permeability of recombinant CP-AMPARs, while increasing polyamine-dependent rectification. To examine the effects of GSG1L on native AMPARs, we manipulated its expression in cerebellar and hippocampal neurons. Transfection of GSG1L into mouse cultured cerebellar stellate cells that lack this protein increased the inward rectification of mEPSCs. Conversely, shRNA-mediated knockdown of endogenous GSG1L in rat cultured hippocampal pyramidal neurons led to an increase in mEPSC amplitude and in the underlying weighted mean single-channel conductance, revealing that GSG1L acts to suppress current flow through native CP-AMPARs. Thus, our data suggest that GSG1L extends the functional repertoire of AMPAR auxiliary subunits, which can act not only to enhance but also diminish current flow through their associated AMPARs.
UNASSIGNED: Calcium-permeable AMPA receptors (CP-AMPARs) are an important group of receptors for the neurotransmitter glutamate. These receptors contribute to various forms of synaptic plasticity, and alterations in their expression or regulation are also seen in a number of serious neurological conditions, including stroke, motor neuron disease, and cocaine addiction. Several groups of auxiliary transmembrane proteins have been described that enhance the function and cell-surface expression of AMPARs. We now report that the recently identified auxiliary protein GSG1L decreases weighted mean channel conductance and calcium permeability of CP-AMPARs while increasing polyamine-dependent rectification by diminishing outward current. Our experiments reveal that GSG1L is an auxiliary subunit that can markedly suppress CP-AMPAR function, in both recombinant systems and central neurons.

This investigation was conducted to study the relationship between intracellular Ca(2+) and activation of large conductance Ca(2+)-activated K(+) (BK) currents by unoprostone, the first synthetic docosanoid. We used HEK293 cells stably transfected with two BK channel splice variants, one sensitive to unoprostone and the other insensitive. We examined the effects of unoprostone on channel activity in excised inside-out patches and cell-attached patches. The half-maximal stimulation of the sensitive BK channels by Ca(2+) was shifted from 3.4±0.017 nM to 0.81±.0058 nM in the presence of 10 nM unoprostone. There was no effect on insensitive channels even at unoprostone concentrations as high as 1000 nM. There was no effect of unoprostone on the voltage dependence of the BK channels. Changes in open probability and effects of Ca(2+) and unoprostone were best described by a synergistic binding model. These data would suggest that Ca(2+) and unoprostone were binding to sites close to one another on the channel protein and that unoprostone binding causes the affinity of the calcium binding site to increase. This idea is consistent with three dimensional models of the Ca(2+) binding site and a putative unoprostone binding domain. Our results have important implications for the clinical use of unoprostone to activate BK channels. Channel activation will be limited if intracellular Ca(2+) is not elevated.