recognition of the importance of ion channel regulation by phosphoinositides ( pips ) , 
 especially phosphatidylinositol 4,5-bisphosphate ( ptdins(4,5)p2 ) , continues to 
 grow . in earlier studies ,
the effects of 
 ptdins(4,5)p2 application were examined directly , or a ptdins(4,5)p2 
 antibody , ptdins(4,5)p2 scavenger ( poly - l - lysine ) or a pharmacological 
 inhibitor ( wortmannin or quercetin ) was used to reduce the membrane ptdins(4,5)p2 
 content .
these methods remain solid and are well established , but their utility is limited 
 to assessing steady - state ptdins(4,5)p2 levels . by contrast , methods for using a 
 chemically inducible pip control system and voltage - sensing phosphatase ( vsp ) are becoming a 
 standardized means of surveying dynamic pip regulation .
chemical induction of pips is well 
 described in a recent review article . in 
 this addendum , therefore , we have focused on the advantages of using vsp to study channel 
 regulation .
so far vsps from two aquatic species , ciona intestinalis ( ci ) and 
 danio rerio ( dr ) , have been identified .
ci- and drvsp exhibit only small 
 differences , mainly in their voltage - sensitivities and expression levels , and their 
 catalytic activities ( pi5-phosphatase ) and substrate phosphoinositide ( pi ) specificities are 
 nearly identical .
nonetheless , we think 
 that drvsp may be somewhat better suited for studying pi - mediated regulation of ion 
 channels .
this is because gating currents indicate the level of drvsp expression in hek 
 cells to be two to three times higher than that of civsp , and because the 
 voltage - sensitivity of drvsp is shifted rightward by about 50 mv 
 ( v12 values from the q - v 
 curves are 94 and 44 mv for drvsp and civsp , respectively ) .
considering that the resting membrane potential is around 40 to 
 10 mv in hek cells , the enzymatic phosphatase activity driven by 
 the resting potential is more negligible with drvsp .
in addition , the rather steep 
 q - v curve for drvsp would be expected to yield more 
 dramatic effects .
when we combined drvsp with trpc channels in hek cells , a depolarizing 
 pulse to + 100 mv for 500 ms was sufficient to produce maximum channel inhibition through 
 depletion of ptdins(4,5)p2 ( referred to as vmi , vsp - mediated inhibition was 
 evaluated based on the residual current ( r ) before and after the 
 depolarization ) . however
, such robust depolarizations can have unfavorable effects on 
 channel regulation , e.g. , the voltage - dependent inhibition relief observed with high 
 voltage - gated ca channels . to exclude this 
 possibility , we confirmed our results using various voltage - sensing drvsp derivatives [ vmi 
 ( r)-v curve ] , as shown in figure  1a .
note that the v - shifted drvsps all inhibited trpc 
 currents to the same degree as wild - type drvsp .
these data support our conclusion and 
 further emphasize the advantages of using drvsp to regulate the activities of a variety of 
 ion channels , including voltage - gated channels .
( a ) top : exemplar of the 
 voltage - dependence of vmi ( r ) of trpc3/c6/c7 currents observed in hek 
 cells .
trpc6 currents were evoked by external application of the dag lipase inhibitor 
 rhc80267 ( 100 m ) .
bottom : vmi of trpc6 currents plotted against depolarization pulse 
 amplitude applied in the presence of the indicated drvsp mutants ( n = 
 more than 4 ) .
note that the q - v curves of the mutants 
 are also shifted leftward [ v1/2(off ] values for 
 r153q , t156r and i165r are 16 , 73 and 60 mv , respectively ) .
( b ) top : atypical inhibition trace obtained from hek 
 cells co - transfected with trpc6 and wild - type drvsp .
brief depolarizations ( + 100 mv , 500 
 ms ) were applied every 10 sec ( protocol displayed in top ) , and currents were evoked by 
 cch ( 100 m ) .
middle and bottom : r and -recovery are plotted against 
 stimulus number from the upper trace .
the blue dashed line in the middle panel suggests 
 drvsp - available ptdins(4,5)p2 ( speculative ) .
the typical trace , averaged 
 r , and averaged -recovery data were shown in ref .
vsp has enormous potential for use as a molecular tool with which to clarify the 
 ptdins(4,5)p2 sensitivity and binding kinetics of ion channels , even during 
 periods when ionic currents are flowing . in figure  1b
, 
 we present an atypical example of the inhibition of carbachol ( cch)-induced trpc6 currents 
 ( upper ) via vsp activation and subsequent current recovery .
in contrast to the typical and 
 averaged data presented in our recent study , in 2 of 11 cells tested , we observed responses like those depicted 
 in the figure [ i.e. , there was a clear bell- or u - shaped relationship between stimulus 
 number and vmi ( r ) ( middle ) and -recovery from the inhibition ( bottom ) ] . 

the bell - shaped r curve was closely related to the peak cch - induced 
 current .
moreover , -recovery accelerated as the cch - induced current and the value of 
 r became larger .
this observation raises the possibility that even during 
 agonist - stimulated macroscopic activity in living cells , vmi magnitude may provide a clue to 
 the level of ptdins(4,5)p2 binding to the channels , as well as to the kinetics of 
 evoked changes in ptdins(4,5)p2 binding .
the latter would also reflect to some 
 degree ptdins(4,5)p2 re - synthesis , which would be expected to influence the 
 observed response . in an earlier study , hardie et al .
, showed dynamics of living 
 ptdins(4,5)p2 which was accompanied with the light response in drosophila 
 photoreceptors by measuring currents through the ptdins(4,5)p2-sensitive kir2.1 
 channel .
however , we suggest the use 
 of ectopic vsp is an alternative or a more convenient approach because vsp does not itself 
 produce ionic flow other than gating currents .
because bioelectric signals are largely attributable to the flow of ions , it is critically 
 important to maintain ion channel activities for appropriate durations . to shorten the 
 duration of ion flow , channels , particularly those contributing to excitation , often possess 
 self - limiting regulatory systems .
for instance , figure  2a to 
 c illustrate the mechanisms of self - limiting regulation found in voltage - gated 
 sodium channels , high voltage - gated calcium channels and inotropic atp receptors .
the 
 mechanism underlying the self - limiting regulation of trpc3/c6/c7 channels ( fig . 
trpc3/c6/c7 channels are intracellular ligand - gated channels assembled as homo- or 
 heterotetramers , and are activated by dag produced through a reaction catalyzed by g 
 protein- or receptor tyrosine kinase - coupled plc .
but when dag is produced from its 
 substrate ptdins(4,5)p2 , the resultant reduction in membrane 
 ptdins(4,5)p2 content independently inhibits channel activation .
thus both 
 activation and inhibition are simultaneously induced by plc - catalyzed degradation of 
 ptdins(4,5)p2 . as a result ,
the time required for the response to reach the 
 first current peak is more susceptible to modulation than the other modes of self - limiting 
 regulation .
( a ) : voltage - gated sodium channels 
 ( rnav1.2 ) in vertebrates open ( + ) and quickly inactivate (  ) in response to 
 depolarization ( 1 and 2 : difference in the potentials coordinate 
 the v - shaped current trace ) .
( b ) : high voltage - gated ca channels ( cav1.2 ) 
 open upon membrane depolarization ( 1 ) and inactivate due to negative 
 feedback regulation wherein ca permeating through the channels inactivates 
 the channels ( calcium - dependent inactivation ) .
( c ) : ligand - gated cys - loop receptors 
 ( p2x1 ) are activated and then desensitized by the concentration - dependent binding of an 
 agonist ( a ) .
copyright ( 1996 ) national academy of sciences , usa ( d ) : trpc3/c6/c7 
 channels are activated by dag , a product of ptdins(4,5)p2 , and the resultant 
 reduction in ptdins(4,5)p2 independently inhibits channel opening .
strength 
 linkages with g protein - coupled receptors alter the kinetics in trpc6 currents ( tight 
 and loose linkages are represented by the red and black traces , respectively ) .
( e ) : currents obtained under 
 self - limiting conditions exhibit faster inactivation than those obtained under unlocked 
 conditions induced by application of excessive ptdins(4,5)p2 through the 
 patch - pipette ( trpc7 currents obtained from ref .
trpc channels are often activated by neurohormones released from autonomic nerves and can 
 be thought of as being downstream of the autonomic nervous system ( ans ) .
the ans is 
 important for maintenance of the stable internal physiological conditions often referred to 
 as  homeostasis .
 this makes the self - limiting regulation of trpc channels interesting in 
 part because the resultant regulation of the global effects of the ans appears to arise from 
 the molecular level .
enzymatic reactions are often involved in cell signaling through , for example , an increase 
 in the concentration of a product . on the other hand ,
the functionality of substrate 
 reduction or depletion is easily masked by the reduction in the productivity of the 
 catalytic reaction .
consequently , signaling systems mediated by enzymatic reactions might be 
 expected to exhibit physiologically bimodal biochemical responses .
the self - limiting 
 regulation linked to plc activity could thus provide profound mechanistic insight of channel 
 modulation as well as rediscovering subtle feature of enzymatic functionality .
furthermore , 
 when this regulation is disrupted ( which can be seen experimentally in the case of 
 cch - induced currents in the presence of an excess of the substrate analog 
 dic8-ptdins[(4,5)p2 ] , the decay phase of the currents is prolonged ( fig . 
2b ) , and the ensuing buildup of cytosolic 
 na and/or ca can have significant pathophysiological effects that 
 could underlie the development of such ailments as vascular hypertension , cardiac 
 hypertrophy and renal failure .
overall , our results emphasize that trpc3/c6/c7 channels are subject to self - limiting 
 regulation that is related to their close association with the 
 ptdins(4,5)p2-plc - dag cascade and is distinct from the self - limiting mechanisms 
 observed in voltage - gated channels .
the specific physiological importance of this type of 
 regulation , as compared with other modes of channel regulation , will be an important area of 
 investigation in the future .
 trpc3/c6/c7 channels , a subgroup of classical / canonical trp channels , are activated by 
 diacylglycerol produced via activation of phospholipase c ( plc)-coupled receptors . 
 
 recognition of the physiological importance of these channels has been steadily growing , 
 but the mechanism by which they are regulated remains largely unknown . 
 we recently used a 
 membrane - resident danio rerio voltage - sensing phosphatase ( drvsp ) to 
 study trpc3/c6/c7 regulation and found that the channel activity was controlled by 
 ptdins(4,5)p2-dag signaling in a self - limiting manner ( imai y et al . , the 
 journal of physiology , 2012 ) . in this addendum , we present the advantages of using drvsp 
 as a molecular tool to study ptdins(4,5)p2 regulation . 
 drvsp should be readily 
 applicable for studying phosphoinositide metabolism - linked channel regulation as well as 
 lipid dynamics . 
 furthermore , in comparison to other modes of self - limiting ion channel 
 regulation , the regulation of trpc3/c6/c7 channels seems highly susceptible to activation 
 signal strength , which could potentially affect both open duration and the time to peak 
 activation and inactivation . 
 dysfunction of such self - limiting regulation may contribute 
 to the pathology of the cardiovascular system , gastrointestinal tract and brain , as these 
 channels are broadly distributed and affected by numerous neurohormonal agonists . 
DrVSP and CiVSP: Time-solved Tool for Channel Regulation
Dynamics of Inhibition and Recovery from the Inhibition by VSP Activation
Self-Limited Ion Channels
Channel Regulation Linked to Enzymatic Reaction