Paragraph Summaries Flashcards
Abstract
We have yet to identify molecules responsible for MA cation channel activities. Characterised a rapidly-adapting MA current in mouse neuroblastoma cell line. Expression profiling and RNA interference knockdown of candidate genes lead to identification of Piezo1 as being required for those currents. Piezos are vertebrate multipass TM proteins w/ homologues in other organisms. Their overexpression induced two kinetically distinct MA currents. Knockdown of Piezo2 in DRG neurons specifically reduced fast-adapting MA currents.
Research proposition/question
Piezos are components of MA cation channels.
Intro
Mechanotransduction is essentially involved in many areas of physiology. Lots of areas of functioning are regulated by mechanotransduction, like pain, touch, proprioception etc in mammals. In plants, morphogenesis. In vertebrates, speed of mechanotransduction in inner-ear hair cells indicates an ion channel directly mechano-activated is involved. Indication of Ca2+ MA cationic currents in different mechanosensitive cells.
Neuro2A cells express MA currents
Seeking a cell line that expresses a MA current similar to those recorded from primary cells. Used a cell culture platform to test the MA current by pressing cultured cells with a glass probe while doing patch-clamp recordings in whole-cell configuration with another pipette. Also, they recorded the single channel currents of N2A cells in response to membrane suction through the recording pipette in cell-attached patch configuration. Through this, they found that mouse neuroblastoma cell line Neuro2A displayed the most consistent MA currents with relatively fast adaptation in response to stimuli.
Piezo1 (Fam38A) is required for MA currents of N2A cells
Looking for candidate ion channels in N2A by looking for transcripts using microarrays.
Requirements for proteins: span membrane ≥ 2X. Narrowed it down to known cation channels or proteins w/ unknown function. Through siRNA knockdown and patch-clamp recordings, found that Fam38A seems important for generation of MA currents in response to stimulation. Had the biggest decrease in MA currents, observed when testing w/ multiple siRNAs that target Fam38A. siRNAs worked in decreasing transcription. Fam38A encodes protein required for expression of pressure-activated ion channels (hence the name Piezo1 because greek). Confirmed that N2A cells can still respond to other stimuli, so the problem is mechanical-specific. Piezo1 might be involved in integrin activation, so they added a buffer to cells that would disrupt integrin function and MA current was not affected, so Piezo1 is not involved in MA currents through integrin function.
Piezos are large-transmembrane proteins conserved among various species
Piezo is found in varying numbers across different species. 2º structure and overall length of Piezo proteins are moderately conserved and have minimal similarity to other proteins. Assays with a special program show that Piezos have between 24 and 36 predicted TM domains located throughout the protein. Piezo 1 is expressed in the bladder, colon, lung, (mechanotransduction related to visceral pain), kidney (mechanotransduction related to urinary flux) and skin. Low amount of Piezo1 in DRGs but strong Piezo2 expression.
Piezo1 induces MA currents in various cell types
Cloned full length Piezo1 into a vector that also encoded GFP and recorded MA currents using patch-clamp 12-48hrs after the vectors were transfected into 3 different cell types – N2A, HEK293T, C2C12. All cells overexpressing Piezo1 had the same current-voltage relationship as endogenous N2A cells. Demonstrated that the ion selectivity of these channels is for cations through 1) blocking channels with blocker which blocked MA currents and 2) Having IC solution containing no cations and adding different combinations of cations to EC solution while measuring MA current (all permeated). Did Assays of Piezo1 transfected cells. Different cell line cells overexpressing Piezo1 were observed to have large currents in response to large negative pressure pulses and current-pressure relationships were similar to endogenous N2A cells, whereas there was no similarity in channel activity between endogenous N2A cells and Piezo1(-) HEK293T cells.
MA currents in cells overexpressing Piezo2
Cloned full-length Piezo2 from DRG neurons into a vector that also encoded GFP. Transfected HEK293T cells with this vector. Cotransfected N2A cells with this vector as well as Piezo1 siRNA to suppress endogenous MA currents, isolating current generated by Piezo2. Piezo2-dependent currents were blocked by NMDG, suggesting nonselective cation conductance. Inactivation kinetics of Piezo2-dependent MA currents were faster than Piezo1-dependent ones for inward and outward currents and for all tested MPs. Conclusion: Piezo1 and Piezo2 confer distinct channel properties.
Piezo1 is detected at the plasma membrane
It looks like the two Piezo proteins are components of the mechanotransduction complexes, so they should be at the PM. Piezo1 has been found in the ER. Transfection with Piezo1 and TRPA1 and staining has indicated that Piezo1 protein can be found at/near PM.
Requirements of Piezo2 for rapidly adapting MA currents in DRG neurons
Performed in situ hybridisation on adult mouse DRG sections to characterize Piezo2 expression within neuron and glial population. Observed its mRNA expression in 20% of DRG neurons. Also found to be expressed in subsets that also express markers for mechanosensory neurons, like peripherin and neurofilament, with some overlap in subsets expressing nociceptive marker TRPV1, so potential involvement in noxious mechanosensation. Transfected DRG neurons with siRNA, scrambled or Piezo2 siRNA, and GFP and recorded whole-cell MA currents from the neurons. The recordings were grouped into 4 groups according to their inactivation kinetics: t < 10ms, 10 < t < 30, t > 30, nonresponsive. The proportion of neurons expressing MA currents with t < 10ms was specifically + significantly reduced in neurons that had been transfected with Piezo2 siRNA. Trend: when transfected with Piezo2 siRNA, see an increased number of mechanically insensitive neurons. Indicates: title.
Discussion
Piezo1 is required for MA currents in N2A cells. Piezo2 is required for a subset of MA currents in DRG neurons. The more you increase their expression, the more-fold increases in MA currents. Large number of predicted TM domains → similar structure to V-gated Na+ channels? But no pore/repetitive domains, so doesn’t seem likely. Piezos could be nonconducting subunits needed for channel expression or modulation. Piezos could also be a distinct class of ion channels. Piezo2 has a potential role for touch and pain sensation. Piezos are expressed in a lot of tissues and homologous in different organisms, so maybe broad role in mechanotransduction.
Conclusion
Piezos are both necessary and sufficient for the expression of a MA current in various cell types.
