Lecture 9: Membrane Function - Regulation of pH and Ca2+

Problem of RMP in Response to Volume Regulation

• RMP (Vm) enhances the gradient for positive ions e.g. H⁺ and Ca²⁺ influx

Challenges of Intracellular Environments

• Difficulting maintaining a narrow biological pH (7.0) to ensure correct protein structure

Enzyme structure

• Influenced by pH - all operate at an optimum pH (most neural)

  • if not at a given/optimum pH can’t function

• Proteins must maintain their 3D structure and can be appropriately changed to function

  • at a low pH can be denatured and change functions

          

pH Of Living Systems

• Operate best within a narrow range of pH (7.0 to 7.6).

Extracellular pH

• ~ 7.4

• Regulated by the lungs and kidney using HCO₃^-/ CO₂ buffering

  • Ventilation makes blood acidic – balances out alkaline substances present in the blood

  • Kidney – concentrates urine and excretes solutes

Intracellular pH

• ~7.2

• Regulated by a combination of membrane transport proteins (active/passive)

  • Lower pH gives rise to the shrinking nature of the cell – actively maintain this relationship with the extracellular environment

Soren Sorensen (1909, Carlsberg Laboratory)

• Defined pH = -log₁₀ [H⁺], when wanting to identify why some beers spoilt

• Was due to pH and its effect on yeast

  • pH must stay within a given range - devation from this compromises yeast function

pH = -log₁₀ [H⁺]

• Useful concept for “chemical” studies of H⁺

  • Logarithmic – ranges from 0 (acidic) to 14 (alkaline)      

• BUT not so helpful in physiological studies where there is a narrow range of [H⁺].

Alternatitve of pH = -log₁₀ [H⁺]

• “pHysiologists” cite [H⁺] in nM.

  • Optimum range = 20 nM to 100 nM

Cardiomyocyte [H⁺]

• At rest 60 nM (7.2)

  • BUT ↑ [H⁺]_i to 100 nM (7.0) causes 50% ↓ contractility - 40nm difference significally affects activity

Challenges to Intracellular pH

• Metabolism: Production of acidic metabolites challenges intracellular pH.

• Ischemia: Reduced blood flow and oxygen supply can lead to increased anaerobic metabolism and acid production.

  • Heart Attack → Stimulation of Acid Sensing Ion Channels (ASIC) = excruciating pain)

• Membrane Transport Proteins: e.g. PMCA and HCO3- channels, can influence intracellular pH. 

pH and Membrane Potential

• A negative membrane potential (Vm) creates an electrochemical gradient that favors the influx of protons (H+).

• This can challenge intracellular pH homeostasis.

• If the membrane potential is 0, the movement of protons will be solely determined by the chemical gradient.

Membrane Potential (Vm) = Equilibrium Potential E_H

• “At equilibrium with Vm”

• Any H⁺ (or any ion) is not actively transported it is said to be distributed at equilibrium

  • EC gradient = 0 so Vm = E_H

Calculation of Intracellular pH:

Typical cell : [H⁺]_o = 40 nM (7.4) and Vm = -60 mV

• -60 = 26.7/1 . ln (40/ [H⁺]_i)

• 60/26.7 = ln (40/ [H⁺]_i)

• e^(-60/26.7) = 40 /[H⁺]_i

• [H⁺]_i  =  40/ e^(-60/26.7)

• [H⁺]_i =  378 nM (6.4) = DEAD

Consequence of Vm = -60mV

• pH has shifted to 6.4

  • Cell cant survive      

• Having a -ve RMP, means cells must actively regulate this      

• Measured [H⁺]_i = 60 nM (7.2), thus, H⁺ must be actively extruded – to increase pH

   

Measuring Intracellular pH

• pH-sensitive fluorescent dyes like BCECF and SNARF can be used to measure intracellular pH.

• These dyes emit light at different wavelengths depending on the pH of their environment.

  • Different spectral sensitivy depending on environemnt → shift from short to long/ higher wavelength

Fluorescent Indicators and Imaging

• Used to measure the concentration of various ions, including calcium (Ca2+) and pH.

• Cells are placed on a microscope stage and illuminated with light of a specific wavelength.

• The emitted light from the fluorescent dye is detected and analyzed to determine the concentration of the ion of interest.

Flurophore

• Developed by Roger Tsien (Nobel Prize 2008)

  • Can measure pH in living cells after calibrating dye  

• along with Ca²⁺ sensitive fluorescent indicators (FURA2-AM “Excitable Cells”) 

Principle of Fluorescence

• When a fluorescent molecule absorbs light energy, it becomes excited.

• As the molecule returns to its ground state, it emits light at a longer wavelength.

• The intensity and wavelength of the emitted light can be used to measure the concentration of the molecule of interest.

Set Up for Measuring pH_i

• Cells present on a microscope stage and is suspended in a bath solution that mimics the concentration of salt/ extracellular environment

• Use conventional light, with a filter or a (confocal) laser to excite the fluroscent dye in the cell

  • Lasers have a specific frequency – monochromatic light

• Excitation of an molecule, shifts an electron to a higher orbit, energy can’t be sustained and so electron is lost and it drops back to its original state

  • Shift is smaller – shift in spectrum – emission of a photon

• Excitation of fluoresce, and place an atom into a different state, ‘as it relaxes’ and drops down to it s original state, to emit a different wavelength e.g. Blue 470nm or a longer wavelength e.g. 600nm

• This can be imaged: increase pH – shift in emission and excitation, and can give rise to a ratio – light in: light out, dependent on concentration of ion

Method Used For Measuring pH_i

• 1. Cells incubated with lipid permeable ester of the indicator

  • Fluorescent molecule has an ester attached – easily crosses membrane - solubilised

• 2. Indicator ester diffuses into cell, is hydrolysed to ionic form and is trapped in cytoplasm

  • Esterase’s cut ester off, trapping dye in the cell

• 3. Indicator is excited      

• 4. Fluorescence is emitted    

• 5. Calibrate using known pH concentrations→ pH_i  

  • Can measure intensity of light to generate a calibration curve – use to measure fluorescence in any condition      

Measure pH_i at equilibrium:

• Useful but does not give much data on underlying mechanisms

        

Measure ΔpH_i when equilibrium is disturbed

• Useful - reveals the mechanism!

Acidification of Cells

• Cells maintain a specific pH range, around 7.4. (Goldilocks period)

• Adding an alkaline solution like NH4+ can disrupt this pH balance, leading to intracellular acidification.

  • raise intracellular pH inresponse to challenge

• Cells have mechanisms to counteract this acidification and restore normal pH levels.

  • Rebound effect

Acidification of Cells: Rebound Effect

• Occurs following the removal of the challenge, where cells adjust their intracelluar pH and returin to normal

• Cell detect there pH above the baseline, equilibrate this and return it to normal

• This can be investiaged through ion substitiuion an/or use of ion transport inhibitors

Acidification of Cells: Investigation of Transport/ Pump

• Use specific inhibitors to block mechanisms (H+/HCO3- transporter) and observe the effects on pH.

  • EIPA can be used to block the H+/HCO3- exchanger.

• Blocking this exchanger can prevent cells from regulating their pH, potentially leading to cell death.

EPIA

• An inhibitor that blocks the H+/HCO3- exchange

  • H+ influx/ HCO3- extrusion mechanism used to regulate pH → causes a decrease in pH

Acid Loaders

• Acitivated in response to alkalisation, high pH

  • Antiporter (CHE exchanger)

  • Co-transporter (CBE)

Acid Extrusion

• Activated in response to acidification, low pH

  • Antiporter (NHE exchanger)

  • Co-transporter (NBC)

    • remove protons in pH TOO low

Mechanisms Used By Cells to Deal With Different pH

• Proteins used as a buffer - charged molecules

  • Negative protein cancels out H+      

• Most cells exist in the Goldilocks zone

Aim of Acid Loaders Extruders

• Aim to maintain goldilocks zone

  • Slow regulation process

Golidlocks Zone

• Permissive pH range/zone: permits biological processes to take place optimally

  • First calibrated by R.V Jones

Reason for Low [Ca²⁺]

• No well tolerated by cells

• A key for intracellular signalling – ubiquitous signal so must be kept at low levels due to signalling action and biological action

Role of Phosphate in Low [Ca²⁺]

• Ca2+ maintained at low levels due to phosphate ions        

• Intracellular concentration = mM range (e.g. free = 0.8 mM)      

• Phosphate readily forms an insoluble precipitate with Ca²⁺ (Calcium-Phosphate bone - insoluble)

• Pathology – calcification of soft tissues

Mechanisms Used to Keep [Ca²⁺]_i extremely low

• Extrusion across cell membrane

• Sequestration into cells

  • Work together to maintain a controlled environment

Ca²⁺ Extrusion

• Na+/Ca2+ antiport exchanger  - 1 Ca2+ out for 3Na+ in      

• Ca2+/ Proton exchanger – 1 Ca2+ out for 2H+ in

  • Uses ATP energy

  • Sometimes 1 Ca2+ out for 1H+ in – but is rare

Sequestration of Ca^2+: ER/SER

• Organelles occupies a large amount of the cytoplasmic space

  • Widely distributed across dendrites and axons

• Regulate signalling and communication through the calcium activity within this compartment of the cell

  • Ca2+ enters the ER using ATP and H+ via Ca2+/H+ antiporter

    • Cell becomes more acidic    

• [Ca2+] in SER/ ER is 300uM (HIGH)

Electroneutral

• No net movement of charge

  •  +ve in and +ve out - balanced

Electrogenic

• Net movement of charge generating a small electrical current

  • 2H+ and 2Ca2+ out–charge not balanced

  • (Includes all ion channels)

Calcium Entry into Cells

• Voltage-gated calcium channels

• Receptor-operated channels (NMDA, AMPA)

  • requre glutamate

• Ryanodine receptors

  • store operated channel

• IP3 receptors

  • GCPR receptor

• Store-operated calcium entry

• Calcium leak channels

  • dependent on temporal frequency required

Calcium Regulation

• Calcium is a key signaling molecule in cells.

• Its levels are tightly regulated by various mechanisms, including:

  • Calcium pumps

  • Calcium exchangers

  • Calcium buffers

Local Calcium Signaling

• Calcium signals can be localized to specific regions of the cell.

• This allows for precise control of cellular processes.

• Calcium buffers help to shape calcium signals and prevent them from spreading too widely

  • Off switch - extrude or sequester

Ca2+ As A Universal Messenger

• A unique electrical-to-chemical signal

• A ubiquitous signal that controls several physiological processes

  • Exocytosis (neurotransmitter release), contraction, enzyme activity, cell division, fluid secretion, cell death, etc.

How Does Ca2+ As A Universal Messger = Different Response

• Temporal Differences in Signaling

  • The same signal can trigger different responses depending on the timing of the signal.

  • For example, calcium signaling can vary in speed and pattern, leading to different cellular outcomes.

• Activation of different signalling pathways

  • entry of Ca2+ via AP can be used as a proxy of electrical activty

Ca2+ in AD Disease Mechanism

• Brain unable to deal with Ca2+

• Not used as a normal signalling molecule

  • Can cause cognitive decline and cell death

Spatial Difference of Ca2+ As A Universal Messenger

• Calcium signals can be localized to specific areas of a cell, such as lipid rafts.

Contraction of Cardiac Muscles

• Calcium ions can propagate through a cell in waves, coordinating cellular processes like muscle contraction.

  • Waves help coordinate contraction across the entire heart

Measurement of Intracellular [Ca2+]

• Developed by Roger Tsien (Nobel Prize 2008)

  • along with Ca²⁺sensitive fluorescent indicators (FURA2-AM)

• Use imaging to measure concentration

  • Ratio can be calculated to detect influx/ extrusion of Ca2+

• Cell flashes every time an action poteinal is generation - due to the influx of Ca2+ - detected by fluorescent molecules

Use of FURA In Ca2+ Measurement

• A calcium-sensitive dye.

• When calcium ions bind to FURA, it fluoresces at 340nm.

• No calcium, FURA fluoresces at 380nm.

• By measuring the change in fluorescence intensity at 340nm and 380nm, we can determine the direction of calcium movement.

Calcium as a Proxy for Electrical Activity

• Calcium influx often accompanies electrical activity in cells, so measuring calcium changes can provide information about electrical signaling.

Genetically Encoded Ca2+ Indicators (GCI)

• indicators in living mice may be used – rats engineered to express a particular protein

• GFP based proteins that detects concentrations of Ca2+ based on the brightness of

• Can use a miniscope attached to mice head to visualise and measure Ca2+ in real life

  • Can analyse the pattern of behaviour and signalling amongst neurons

Cellular Homeostasis

• Cell volume, intracellular pH, intracellular calcium, and membrane potential are crucial for cellular function.

  • Parameters are interconnected and influence each other.

• Understanding these interrelationships is essential for studying cellular function experimentally.