Radhakrishnan Gnanasambandam

Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1

Significance Familial xerocytosis in humans, which causes dehydration of red blood cells and hemolytic anemia, was traced to mutations in the mechanosensitive ion channel, PIEZO1. The mutations slowed inactivation and introduced a pronounced latency for activation. Loss of inactivation and increased latency for activation could modify groups of channels simultaneously, suggesting that they exist in common spatial domains. The hereditary xerocytosis mutants affect red cell cation fluxes: slow inactivation increases them, and increased latency decreases them. These data provide a direct link between pathology and mechanosensitive channel dysfunction in nonsensory cells. Familial xerocytosis (HX) in humans is an autosomal disease that causes dehydration of red blood cells resulting in hemolytic anemia which has been traced to two individual mutations in the mechanosensitive ion channel, PIEZO1. Each mutation alters channel kinetics in ways that can explain the clinical presentation. Both mutations slowed inactivation and introduced a pronounced latency for activation. A conservative substitution of lysine for arginine (R2456K) eliminated inactivation and also slowed deactivation, indicating that this mutant’s loss of charge is not responsible for HX. Fitting the current vs. pressure data to Boltzmann distributions showed that the half-activation pressure, P1/2, for M2225R was similar to that of WT, whereas mutations at position 2456 were left shifted. The absolute stress sensitivity was calibrated by cotransfection and comparison with MscL, a well-characterized mechanosensitive channel from bacteria that is driven by bilayer tension. The slope sensitivity of WT and mutant human PIEZO1 (hPIEZO1) was similar to that of MscL implying that the in-plane area increased markedly, by ∼6–20 nm2 during opening. In addition to the behavior of individual channels, groups of hPIEZO1 channels could undergo simultaneous changes in kinetics including a loss of inactivation and a long (∼200 ms), silent latency for activation. These observations suggest that hPIEZO1 exists in spatial domains whose global properties can modify channel gating. The mutations that create HX affect cation fluxes in two ways: slow inactivation increases the cation flux, and the latency decreases it. These data provide a direct link between pathology and mechanosensitive channel dysfunction in nonsensory cells.

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels.

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35–55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.

Hereditary xerocytosis revisited

A 21 year old male student presented in 1980 as an Olympic athlete with a 12 year history of jaundice, pallor, and darkened urine induced by the atraumatic exercise of swimming (1). Physical examination at that time was remarkable only for moderate scleral icterus without hepatosplenomegaly. Hematological examination revealed moderate macrocytosis (MCV 102 fL) without anemia (Hct 50%, Hb 17 g/dL, 9% reticulocytes). The peripheral blood smear showed occasional target cells. Red cell osmotic fragility was decreased. Red cell Na content was increased and K content was decreased, with reduced total monovalent ion content. Passive red cell permeability of both Na and K were increased. A supervised 2.5 hr swimming workout increased free plasma Hb from <5 to 45 mg/dL and decreased serum haptoglobin from 25 to 6 mg/dL. The post-exercise urine sediment was remarkable for hemosiderin-laden tubular epithelial cells, without frank hemoglobinuria. The circulating 15 day erythrocyte half-life measured after 6 days without exercise was further shortened to 12 days after resumption of twice-per-day swimming workouts for 1 week. The patient’s red cells were hypersensitive to in vitro shear stress applied by cone-plate viscometer.

Adaptation Independent Modulation of Auditory Hair Cell Mechanotransduction Channel Open Probability Implicates a Role for the Lipid Bilayer

The auditory system is able to detect movement down to atomic dimensions. This sensitivity comes in part from mechanisms associated with gating of hair cell mechanoelectric transduction (MET) channels. MET channels, located at the tops of stereocilia, are poised to detect tension induced by hair bundle deflection. Hair bundle deflection generates a force by pulling on tip-link proteins connecting adjacent stereocilia. The resting open probability (Popen) of MET channels determines the linearity and sensitivity to mechanical stimulation. Classically, Popen is regulated by a calcium-sensitive adaptation mechanism in which lowering extracellular calcium or depolarization increases Popen. Recent data demonstrated that the fast component of adaptation is independent of both calcium and voltage, thus requiring an alternative explanation for the sensitivity of Popen to calcium and voltage. Using rat auditory hair cells, we characterize a mechanism, separate from fast adaptation, whereby divalent ions interacting with the local lipid environment modulate resting Popen. The specificity of this effect for different divalent ions suggests binding sites that are not an EF-hand or calmodulin model. GsMTx4, a lipid-mediated modifier of cationic stretch-activated channels, eliminated the voltage and divalent sensitivity with minimal effects on adaptation. We hypothesize that the dual mechanisms (lipid modulation and adaptation) extend the dynamic range of the system while maintaining adaptation kinetics at their maximal rates. SIGNIFICANCE STATEMENT Classically, changes in extracellular calcium and voltage affect open probability (Popen) through mechanoelectric transduction adaptation, and this mechanism is the only means of controlling the set point of the channel. Here, we further characterize the effects of extracellular calcium and voltage on the channel and for the first time determine that these manipulations occur through a mechanism that is independent of fast adaptation and involves the lipid bilayer. These data additionally demonstrate that effects on Popen are not enough to characterize adaptation and thus may clarify some of the discrepancies within the literature as to mechanisms underlying adaptation.

Silencing of Kir2 channels by caveolin-1: cross-talk with cholesterol

Inwardly rectifying potassium channels (Kir) play key roles in regulating membrane excitability and K+ homeostasis in multiple cell types. Our earlier studies showed that Kir2 channels, one of the major subfamilies of Kir, are suppressed by membrane cholesterol and that cholesterol stabilizes these channels in a closed ‘silent’ state. This paper addresses a fundamental question of how Kir2 channels are regulated by caveolins, the major structural proteins of caveolae, and the relationship between the sensitivity of the channels to caveolin and to cholesterol. In this study, we present direct evidence that caveolin‐1 is a negative regulator of Kir2 function and that cholesterol and caveolin‐1 regulate the channels by a common mechanism. This study also challenges a general notion that cholesterol depletion alters ion channel function by disrupting caveolae, demonstrating that neither caveolin‐1 nor intact caveolae are required for cholesterol sensitivity of Kir2 channels. Furthermore, we present first insights into the structural determinants of the cross‐talk between the sensitivity of Kir2 channels to caveolin and to cholesterol.

Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations.

GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5e) due to six Lys residues. The peptide does not have a stereospecific binding site on the channel but acts from the boundary lipids within a Debye length of the pore probably by changing local stress. To gain insight into how these Lys residues interact with membranes, we performed molecular dynamics simulations of Lys to Glu mutants in parallel with our experimental work. In silico, K15E had higher affinity for 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine bilayers than wild-type (WT) peptide or any other mutant tested, and showed deeper penetration than WT, a finding consistent with the experimental data. Experimentally, the inhibitory activities of K15E and K25E were most compromised, whereas K8E and K28E inhibitory activities remained similar to WT peptide. Binding of WT in an interfacial mode did not influence membrane thickness. With interfacial binding, the direction of the dipole moments of K15E and K25E was predicted to differ from WT, whereas those of K8E and K28E oriented similarly to that of WT. These results support a model in which binding of GsMTx4 to the membrane acts like an immersible wedge that serves as a membrane expansion buffer reducing local stress and thus inhibiting channel activity. In simulations, membrane-bound WT attracted other WT peptides to form aggregates. This may account for the positive cooperativity observed in the ion channel experiments. The Lys residues seem to fine-tune the depth of membrane binding, the tilt angle, and the dipole moments.

Mechanical stress activates NMDA receptors in the absence of agonists

While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca2+ entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca2+ influx. Extracellular Mg2+ at 2 mM did not significantly affect the shear induced Ca2+ influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.

Unsupervised Idealization of Ion Channel Recordings by Minimum Description Length: Application to Human PIEZO1-Channels

Researchers can investigate the mechanistic and molecular basis of many physiological phenomena in cells by analyzing the fundamental properties of single ion channels. These analyses entail recording single channel currents and measuring current amplitudes and transition rates between conductance states. Since most electrophysiological recordings contain noise, the data analysis can proceed by idealizing the recordings to isolate the true currents from the noise. This de-noising can be accomplished with threshold crossing algorithms and Hidden Markov Models, but such procedures generally depend on inputs and supervision by the user, thus requiring some prior knowledge of underlying processes. Channels with unknown gating and/or functional sub-states and the presence in the recording of currents from uncorrelated background channels present substantial challenges to such analyses. Here we describe and characterize an idealization algorithm based on Rissanens Minimum Description Length (MDL) Principle. This method uses minimal assumptions and idealizes ion channel recordings without requiring a detailed user input or a priori assumptions about channel conductance and kinetics. Furthermore, we demonstrate that correlation analysis of conductance steps can resolve properties of single ion channels in recordings contaminated by signals from multiple channels. We first validated our methods on simulated data defined with a range of different signal-to-noise levels, and then showed that our algorithm can recover channel currents and their substates from recordings with multiple channels, even under conditions of high noise. We then tested the MDL algorithm on real experimental data from human PIEZO1 channels and found that our method revealed the presence of substates with alternate conductances.

Effect of Inflammatory Mediators on Cytoskeletal Stress and Endogenous Mechanosensitive Currents in Dorsal Root Ganglion Neurons

Mechanosensitive channel (MSC) activity elicited by stretching membrane patches or by cell indentation is tightly coupled to the state of the cytoskeleton. To determine how stress in the cytoskeleton changes in the presence of inflammatory mediators we transiently expressed multiple cpstFRET probe chimeras (actinin, filamin and spectrin) in dorsal root ganglion (DRG) neurons and measured their stress responses in the indentation assay. PGE2 significantly increased both the resting stress and the stress response upon indentation in multiple cytoskeletal chimeras. PGE2 induced a robust increase in stress in filamin probes in the indentation assay. In the same assay, CGS-21680 induced a moderate increase in stress whereas 5-HT (serotonin) did not influence stress. The stress change profile resulting from the application of the above-mentioned inflammatory mediators (PGE2, CGS-21680 and 5-HT) correlated with the changes in endogenous mechanosensitive currents elicited by indenting DRG neurons. PGE2 increased mechanosensitive current substantially whereas only moderate increases were observed for CGS-21680 and 5-HT.

Properties of Human Pieoz1 Bearing Two Mutations Identified in Xerocytosis

Piezo1 is a cation selective channel isolated from eukaryotes which shows voltage dependent inactivation. Xerocytosis is a condition that causes hemolytic anemia of red blood cells associated with either of two individual mutations in the gene encoding Piezo1. In this work we have incorporated both mutations together into the Piezo1 gene and characterized the properties of the new channel. The protein has a substitution of arginine for methionine at position 2225 (M2225R) and a lysine for arginine at position 2455 (R2455K). The double mutant showed no voltage dependent inactivation, a property also seen for the single mutant R2455K. We measured the pressure dependence of the channel in cell attached mode and found it to be shifted significantly leftward (Boltzmann). The ability to activate Piezo1 at low pressure has allowed us to determine more accurately the kinetics of Piezo1 channel gating. The conductance of the double mutant is ∼ 30 pS. Whole cell currents were significantly larger, indicating that the double-mutant channel is more sensitive to cell mechanical stimulation. We have co-transfected Piezo1 and MscL and sequentially activated both channels with increasing pressure, allowing us to compare area changes within a single patch. The double mutated channel appears to have changes similar to those found for MscL. We also show that the channel is blocked by the addition of GsMTx4, a peptide inhibitor for mechanical channels.