Surface charge near the cardiac inward-rectifier channel measured from single-channel conductance

Summary The conductance of a channel to permeable ions depends on the number of ions near the mouth of the pore. Surface charge controls the local concentration, and impermeable cations can modify this charge. Correlating channel conductance with the concentration of impermeable cations therefore determines the local charge near the open pore. This paper presents data from cell-attached patches on embryonic chick ventricle cells, and it uses the conductance of inward-rectifier channels in the patch (in 100mm K, with various concentrations of Na, Ca, Ba, and Mg) to estimate the local surface potential. The results indicate the presence of ionized residues near the mouth of the channel. Using the Boltzmann equation and the Gouy-Chapman relation, the surface potential due to these residues (in 100K/33Na/0Ca/0Ba/0Mg) is –40 mV, and the charge density is –0.25e/nm².     

Mechanism of generation of spontaneous miniature outward currents (SMOCs) in retinal amacrine cells

A subtype of retinal amacrine cells displayed a distinctive array of K(+) currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of -60 to -40 mV. Depolarizations above approximately -40 mV were associated with the disappearance of SMOCs and the appearance of transient (I(to)) and sustained (I(so)) outward K(+) currents. I(to) appeared at about -40 mV and its apparent magnitude was biphasic with voltage, whereas I(so) appeared near -30 mV and increased linearly. SMOCs, I(to), and a component of I(so) were Ca(2+) dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of -20 mV and above. Their mean amplitude was Nernstian with respect to [K(+)](ext) and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca(2+)-free solutions, indicative of BK-type Ca(2+)-activated K(+) currents. Dihydropyridine Ca(2+) channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca(2+) permeation through the kainic acid receptor had no effect. Blockade of organelle Ca(2+) channels by ryanodine, or intracellular Ca(2+) store depletion with caffeine, eradicated SMOCs. Internal Ca(2+) chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca(2+) influx through L-type Ca(2+) channels and its subsequent amplification by Ca(2+)-induced Ca(2+) release via the ryanodine receptor leads to a localized elevation of internal Ca(2+). This amplified Ca(2+) signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.     

Ca2+-activated chloride channel activity during Ca2+ alternans in ventricular myocytes

Cardiac alternans, defined beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias. We investigated mechanisms of cardiac alternans in single rabbit ventricular myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. A strong correlation between beat-to-beat alternations of AP morphology and CaT alternans was observed. During CaT alternans application of voltage clamp protocols in form of pre-recorded APs revealed a prominent Ca²⁺-dependent membrane current consisting of a large outward component coinciding with AP phases 1 and 2, followed by an inward current during AP repolarization. Approximately 85% of the initial outward current was blocked by Cl^? channel blocker DIDS or lowering external Cl^? concentration identifying it as a Ca²⁺-activated Cl^? current (I_CaCC). The data suggest that I_CaCC plays a critical role in shaping beat-to-beat alternations in AP morphology during alternans.     

Mitochondrial Ca2+-activated K+ channels and their role in cell life and death pathways

Ca²⁺-activated K⁺ channels (K_Ca) are expressed at the plasma membrane and in cellular organelles. Expression of all K_Ca channel subtypes (BK, IK and SK) has been detected at the inner mitochondrial membrane of several cell types. Primary functions of these mitochondrial K_Ca channels include the regulation of mitochondrial ROS production, maintenance of the mitochondrial membrane potential and preservation of mitochondrial calcium homeostasis. These channels are therefore thought to contribute to cellular protection against oxidative stress through mitochondrial mechanisms of preconditioning. In this review, we summarize the current knowledge on mitochondrial KCa channels, and their role in mitochondrial function in relation to cell death and survival pathways. More specifically, we systematically discuss studies on the role of these mitochondrial K_Ca channels in pharmacological preconditioning, and according protective effects on ischemic insults to the brain and the heart.     

Calcium-activated K+ channels: Metabolic regulation

Calcium-activated potassium (K_Ca) channels are highly modulated by a large spectrum of metabolites. Neurotransmitters, hormones, lipids, and nucleotides are capable of activating and/or inhibiting K_Ca channels. Studies from the last few years have shown that metabolites modulate the activity of K_Ca channels via: (1) a change in the affinity of the channel for Ca²⁺ (K_1/2 is modified), (2) a parallel shift in the voltage axis of the acitvation curves, or (3) a change in the slope (effective valence) of the voltage dependence curve. The shift of the voltage dependence curve can be a direct consequence of the change in the affinity for Ca²⁺. Recently, the mechanistic steps involved in the modulation of K_Ca channels are being uncovered. Some interactions may be direct on K_Ca channels and others may be mediated via G-proteins, second messengers, or phosphorylation. The information given in this review highlights the possibility that K_Ca channels can be activated or inhibited by metabolites without a change in the intracellular Ca²⁺ concentration.     

Upregulation of KCNQ1/KCNE1 K+ channels by Klotho

Klotho is a transmembrane protein expressed primarily in kidney, parathyroid gland, and choroid plexus. The extracellular domain could be cleaved off and released into the systemic circulation. Klotho is in part effective as β-glucuronidase regulating protein stability in the cell membrane. Klotho is a major determinant of aging and life span. Overexpression of Klotho increases and Klotho deficiency decreases life span. Klotho deficiency may further result in hearing loss and cardiac arrhythmia. The present study explored whether Klotho modifies activity and protein abundance of KCNQ1/KCNE1, a K⁺ channel required for proper hearing and cardiac repolarization. To this end, cRNA encoding KCNQ1/KCNE1 was injected in Xenopus oocytes with or without additional injection of cRNA encoding Klotho. KCNQ1/KCNE1 expressing oocytes were treated with human recombinant Klotho protein (30 ng/ml) for 24 h. Moreover, oocytes which express both KCNQ1/KCNE1 and Klotho were treated with 10 µM DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. The KCNQ1/KCNE1 depolarization-induced current (I_Ks) was determined utilizing dual electrode voltage clamp, while KCNQ1/KCNE1 protein abundance in the cell membrane was visualized utilizing specific antibody binding and quantified by chemiluminescence. KCNQ1/KCNE1 channel activity and KCNQ1/KCNE1 protein abundance were upregulated by coexpression of Klotho. The effect was mimicked by treatment with human recombinant Klotho protein (30 ng/ml) and inhibited by DSAL (10 µM). In conclusion, Klotho upregulates KCNQ1/KCNE1 channel activity by 'mainly' enhancing channel protein abundance in the plasma cell membrane, an effect at least partially mediated through the β-glucuronidase activity of Klotho protein.     

Upregulation of KCNQ1/KCNE1 K+ channels by Klotho

Klotho is a transmembrane protein expressed primarily in kidney, parathyroid gland, and choroid plexus. The extracellular domain could be cleaved off and released into the systemic circulation. Klotho is in part effective as β-glucuronidase regulating protein stability in the cell membrane. Klotho is a major determinant of aging and life span. Overexpression of Klotho increases and Klotho deficiency decreases life span. Klotho deficiency may further result in hearing loss and cardiac arrhythmia. The present study explored whether Klotho modifies activity and protein abundance of KCNQ1/KCNE1, a K⁺ channel required for proper hearing and cardiac repolarization. To this end, cRNA encoding KCNQ1/KCNE1 was injected in Xenopus oocytes with or without additional injection of cRNA encoding Klotho. KCNQ1/KCNE1 expressing oocytes were treated with human recombinant Klotho protein (30 ng/ml) for 24 h. Moreover, oocytes which express both KCNQ1/KCNE1 and Klotho were treated with 10 µM DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. The KCNQ1/KCNE1 depolarization-induced current (I_Ks) was determined utilizing dual electrode voltage clamp, while KCNQ1/KCNE1 protein abundance in the cell membrane was visualized utilizing specific antibody binding and quantified by chemiluminescence. KCNQ1/KCNE1 channel activity and KCNQ1/KCNE1 protein abundance were upregulated by coexpression of Klotho. The effect was mimicked by treatment with human recombinant Klotho protein (30 ng/ml) and inhibited by DSAL (10 µM). In conclusion, Klotho upregulates KCNQ1/KCNE1 channel activity by 'mainly' enhancing channel protein abundance in the plasma cell membrane, an effect at least partially mediated through the β-glucuronidase activity of Klotho protein.     

Zebrafish heart as a model for human cardiac electrophysiology

The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.     

Ca2+-activated K+ channels from cultured renal medullary thick ascending limb cells: Effects of pH

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb cells (MTAL) using the patch-clamp technique. The purpose was to determine the effect of acidic pH on channel properties in excised patches of apical cell membrane. At pH 7.4, increasing Ca²⁺ on the intracellular side or applying positive voltages increases channel open probability. Reducing pH to 5.8 on the intracellular face of the channel decreases channel open probability at each voltage and Ca²⁺ concentration. Channel mean open times display two distributions and mean closed times display three distributions. Increasing Ca²⁺ or applying depolarizing voltages lengthens each of the mean open times and shortens each of the closed times. Lowering pH to 5.8 decreases the mean open times and increases mean closed times at each Ca²⁺ and voltage with the greatest effect on the mean closed times. In contrast, both single-channel conductance and channel kinetics are unaffected when pH is reduced to 5.8 on the extracellular face of the membrane. We conclude that protons interfere with Ca²⁺ binding to the gate of Ca²⁺-activated K⁺ channels reducing the probability of channel opening.     

Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 microM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 +/- 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 +/- 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2-5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an "IQ-like" motif present in the carboxy terminus of KCNQ2-5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic "gene gun." Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.     

Modulation of the voltage sensor of L-type Ca2+ channels by intracellular Ca2+

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.     

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca²⁺ activation site was modified using N-bromoacetamide (NBA). 1mm NBA in the bath solution, at 2.5 m Ca²⁺ reduces the open probability,P _o , of the channel to <0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca²⁺-sensitive, requiring 25mm Ca²⁺ to raiseP _o to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca²⁺ (1mm) protects the channels from modification. Also presented is a second class of Ca²⁺-activated K⁺ channels which are normally present in MTAL cells which open infrequently at 10 m Ca²⁺ (P _o =0.01) but have aP _o of 0.08 at 1mm Ca²⁺. We can conclude (i) that NBA modifies the channel by shifting Ca²⁺-sensitivity to very high Ca²⁺, (ii) that NBA acts on a site involved in Ca²⁺ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.     

Molecular properties of sodium and calcium channels

Voltage-gated sodium and calcium channels are responsible for inward movement of sodium and calcium during electrical signals in cell membranes. Their principal subunits are members of a gene family and can function as voltage-gated ion channels by themselves. They are expressed in association with one or more auxiliary subunits which increase functional expression and modify the functional properties of the principal subunits. Structural elements which are required for voltage-dependent activation, selective ion conductance, and inactivation have been identified, and their mechanisms of action are being explored through mutagenesis, expression in heterologous cells, and functional analysis. These experiments reveal that these two channels are built on a common structural theme with variations appropriate for functional specialization of each channel type.     

Activation of K+ and Cl− channels in MDCK cells during volume regulation in hypotonic media

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24 +/- 4 pS in symmetrical K+ concentrations (145 mM). It was highly selective for K+ ions vs. Na+ ions: PNa/PK less than 0.04. The time course of its open probability (P0) showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3 +/- 4.7 pS and inward slope conductance of 26.1 +/- 4.9 pS. It was permeable to both Cl- and NO3- and its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 mM quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD.     

Grina/TMBIM3 modulates voltage-gated CaV2.2 Ca2+ channels in a G-protein-like manner

Grina/TMBIM3 is a poorly characterized transmembrane protein with a broad expression pattern in mammals and with a very ancient origin within eukaryotes. Although initially characterized as an NMDA-receptor associated subunit, there is increasing evidence that Grina/TMBIM3 is involved in the unfolded protein response and controls apoptosis via regulation of Ca²⁺ homeostasis. Here, we investigate a putative direct interaction of Grina/TMBIM3 with voltage gated Ca²⁺ channels, in particular with the Ca_V2.2 α1-subunit and describe its modulatory effects on the current through Ca_V2.2 N-type channels. Direct interaction was confirmed by co-immunoprecipitation studies and membrane localization was proven. Co-expression of Grina/TMBIM3 with Ca_V2.2 channels resulted in a significant decrease of the current amplitude and in a slowing of the kinetics of current activation. This effect was accompanied by a significant shift of the voltage dependencies of activation time constants towards more depolarized voltages. Application of a stimulus protocol including a strong depolarizing pulse relieved inhibition of current amplitude by Grina/TMBIM3. When Grina/TMBIM3 was present, inactivation by an action potential-like train of pulses was diminished. Both observations resemble mechanisms that are well-studied modulatory effects of G-protein βγ subunits on Ca_V2 channels. The impact of Grina/TMBIM3 and G-protein βγ subunits are rather comparable with respect to suppression of current amplitude and slowing of activation kinetics. Furthermore, both modulators had the same effect on current inactivation when evoked by an action potential-like train of pulses.     

Tyrosine kinases act directly on the alpha1 subunit to modulate Ca(v)2.2 calcium channels.

Voltage-operated calcium channels are modulated by tyrosine kinases in different cell types. In this study, I(Ba) was measured by the whole cell voltage-clamp technique in single COS-7 cells overexpressing the Ca(v)2.2 calcium channels encoding N-type currents. Bath application of genistein, a nonselective PTK inhibitor (50-300 microM), concentration-dependently inhibited calcium channel currents, whereas the inactive structural analogue, daidzein, was without effect over the same concentration range. Similarly, PP1, a src family-selective tyrosine kinase inhibitor, inhibited I(Ba) in a concentration-dependent manner (500 nM-5 microM) over a range of test potentials. Expression of the Ca(v)2.2alpha1 (alpha(1B)) subunit alone gave rise to functional channels, and genistein (100 microM) also inhibited currents elicited by the alpha(1B) subunit alone. These results indicate that tyrosine kinase inhibitors are likely to inhibit Ca(v)2.2 calcium channels via an action on the pore-forming alpha(1) subunit and suggest that an endogenous member of the Src family may play a physiological role in modulating these channels.